Dedifferentiated, programmable stem cells of monocytic origin, and their production and use

ABSTRACT

The invention relates to the production of adult dedifferentiated, programmable stem cells from human monocytes by cultivation of monocytes in a culture medium which contains M-CSF and IL-3. The invention further relates to pharmaceutical preparations, which contain the dedifferentiated, programmable stem cells and the use of these stem cells for the production of target cells and target tissue.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 120 as acontinuation-in-part application of U.S. application Ser. No.10/372,657, filed Feb. 25, 2003 now abandoned which claims the benefitunder 35 U.S.C. § 119 of German Patent Application No. 102 14 095.2,filed Mar. 28, 2002. This application also claims the benefit under 35U.S.C. § 365 of International Application No. PCT/EP03/02121 filed Feb.25, 2003, which claims the benefit of German Patent Application Number102 14 095.2, filed Mar. 28, 2002. The disclosures of these applicationsare hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The term “stem cells” designates cells which (a) have the capability ofself-renewal and (b) the capability to form at least one and often anumber of specialized cell types due to their asymmetrical divisioncapability (cf. Donovan, P. J., Gearhart, J., Nature 414: 92–97 (2001)).The term “pluripotent” designates stem cells, which can essentially bedifferentiated into all possible cell types of the human and animalbody. Such stem cells have hitherto only been obtainable from embryonictissue or embryonic carcinoma (testicular tumor) (cf. Donovan, P. J.,Gearhart, J., loc cit). The use of embryonic stem cells has been thesubject of extensive public discussion, especially in Germany, and isregarded as extremely problematical. Besides the ethical and legalproblems connected with embryonic stem cells, the therapeutic use ofsuch cells also comes up against difficulties. By nature, embryonic stemcells are obtained from donor organisms, which are heterologousvis-à-vis the potential recipients of differentiated cells or tissue(hereafter referred to as somatic target cells or target tissue)developed from these cells. It is therefore to be expected, that suchtarget cells will trigger an immediate immunological response in thepotential recipients in the form of rejection.

Stem cells can be also isolated from different tissues of adult, i.e.,from differentiated individuals. Such stem cells are referred to in thestate of the art as “multipotent adult stem cells”. In the body theyplay a role in tissue regeneration and homeostasis. The essentialdifference between embryonic pluripotent stem cells and adultmultipotent stem cells lies in the number of differentiated tissues,which can be obtained from the respective cells. Presumably, this is dueto the fact that pluripotent stem cells come from sperm cells, or fromcells which can produce sperm, while adult multipotent stem cells comefrom the body or soma of adult individuals (cf. Donovan, P. J.,Gearhart, J. loc cit, Page 94), which are not capable of spermproduction.

The actual problems relating to the obtaining and use of adult stemcells however lie in the rarity of these cells. Thus, in the bonemarrow, stem cells are present only in the ratio of 1:10,000, in theperipheral blood of 1:250,000 and in the liver in the ratio of1:100,000. Obtaining such stem cells is therefore very expensive andstressful for the patient. In addition the generation of large cellquantities, as required for clinical therapy, has scarcely been possiblehitherto at reasonable expense.

This is contrasted by a constantly increasing need for possibilities fortreatment of destroyed tissue in the form of “tissue engineering” or ascell therapy, within the framework of which skin-, muscle-, heartmuscle-, liver-, islet-, nerve-, neurone-, bone-, cartilage-,endothelium- and fat cells etc. are to be replaced.

In this connection, the foreseeable development of the age and diseaseprofile of the population in the western world is decisive, leading tothe expectation of a drastic turning point in the next 10 years in thehealth and care sector of the western European population, including theUSA and Canada. In the Federal Republic of Germany alone, thedemographic development suggests a 21%-growth in population in the 45–64year-old age group by 2015, and a 26%-growth in the over 65 age group.This is bound to result in a change in patient structure and in thespectrum of diseases requiring treatment. Predictably, diseases of thecardio-circulatory system (high pressure, myocardial infarction),vascular diseases due to arteriosclerosis and metabolic diseases,metabolic diseases such an diabetes mellitus, diseases at livermetabolism, kidney diseases as well as diseases of the skeletal systemcaused by age-related degeneration, and degenerative diseases of thecerebrum caused by neuronal and glial cell losses will increase andrequire innovative treatment concepts.

These facts explain the immense national and international research anddevelopment efforts by the specialists involved, to obtain stem cellswhich can be programmed into differentiated cells typical of tissue(liver, bone, cartilage, muscle, skin etc.).

The problem underlying the invention therefore resides in makingavailable adult stem cells, the generation of which gives rise to noethical and/or legal problems, which are rapidly available for theplanned therapeutic use in the quantities required for this, and atjustifiable production costs, and which, when used as “cellulartherapeutics” give rise to no side effects—or none worth mentioning—interms of cellular rejection and induction of tumors, particularlymalignant tumors, in the patient in question.

SUMMARY OF THE INVENTION

The present invention provides a method for producing humandedifferentiated programmable stem cells using M-CSF and IL-3.

The present invention includes and provides a process for the productionof dedifferentiated, programmable stem cells of human monocytic origin,comprising (a) isolating the monocytes from human blood; (b) propagatingthe monocytes in a culture medium, which contains cellular growth factorM-CSF; (c) simultaneously cultivating the monocytes with or subsequentlyto step (b) in a culture medium comprising IL-3; and (d) obtaining humanadult dedifferentiated programmable stem cells by separating fromculture medium.

The present invention includes and provides a process for the productionof dedifferentiated, programmable stem cells of human monocytic origin,comprising (a) providing human monocytes; (b) propagating the monocytesin a culture medium, which contains cellular growth factor M-CSF; (c)simultaneously cultivating the monocytes with or subsequently to step(b) in a culture medium comprising IL-3; and (d) obtaining human adultdedifferentiated programmable stem cells by separating from culturemedium.

The present invention includes and provides a dedifferentiated,programmable stem cell of human monocytic origin, wherein the cell ischaracterized by exhibiting a CD14 antigen and an antigen selected fromthe group consisting of CD90, CD117, CD123 and CD135.

The present invention includes and provides a dedifferentiated,programmable stem cell of human monocytic origin, wherein the cell ischaracterized by exhibiting a CD14 antigen and a CD123 antigen.

The present invention includes and provides a dedifferentiated,programmable stem cell of human monocytic origin, wherein the cell ischaracterized by exhibiting a CD14 antigen and a CD135 antigen.

The present invention includes and provides a dedifferentiated,programmable stern cell of human monocytic origin, wherein the cell ischaracterized by exhibiting a CD14 antigen, a CD 123 antigen and a CD135 antigen.

The present invention includes and provides a dedifferentiated,programmable stem cell of human monocytic origin manufactured by aprocess comprising (a) isolating monocytes from human blood; (b)propagating monocytes in a culture medium, which contains cellulargrowth factor M-CSF; (c) simultaneously cultivating monocytes with orsubsequently to step (b) in a culture medium comprising IL-3; and (d)obtaining human adult dedifferentiated programmable stem cells byseparating from culture medium.

The present invention includes and provides a pharmaceutical compositioncomprising a dedifferentiated, programmable stem cell of human monocyticorigin, wherein the cell is characterized by exhibiting a CD14 antigenand an antigen selected from the group consisting of CD90, CD117, CD123and CD135.

The present invention includes and provides a pharmaceutical compositioncomprising a dedifferentiated, programmable stem cell of human monocyticorigin, wherein the cell is characterized by exhibiting a CD14 antigenand a CD135 antigen.

The present invention includes and provides a pharmaceutical compositioncomprising a dedifferentiated, programmable stem cell of human monocyticorigin, wherein the cell is characterized by exhibiting a CD 14 antigenand a CD 123 antigen.

The present invention includes and provides a pharmaceutical compositioncomprising a dedifferentiated, programmable stem cell of human monocyticorigin, wherein the cell is characterized by exhibiting a CD14 antigen,a CD123 antigen and a CD135 antigen.

The present invention includes and provides a method of producing targetcells from dedifferentiated, programmable stem cells of human monocyticorigin comprising (a) obtaining desired target cells from a targettissue; (b) incubating the desired target cells in a suitable culturemedium; and (c) providing supernatent from the culture medium afterincubation with the desired target cells to dedifferentiated,programmable stem cells of human monocytic origin that are characterizedby exhibiting a CD 14 antigen and an antigen selected from the groupconsisting of CD90, CD117, CD123 and CD135 to differentiate said stemcells of human monocytic origin into target cells.

The present invention includes and provides a method of producing targetcells from dedifferentiated, programmable stem cells of human monocyticorigin comprising (a) obtaining desired target cells from a targettissue; (b) incubating the desired target cells in a suitable culturemedium; and (c) providing supernatent from the culture medium afterincubation with the desired target cells to dedifferentiated,programmable stem cells of human monocytic origin that are characterizedby exhibiting a CD14 and a CD135 antigen to differentiate said stemcells of human monocytic origin into target cells.

The present invention includes and provides a method of producing targetcells from dedifferentiated, programmable stem cells of human monocyticorigin comprising (a) obtaining desired target cells from a targettissue; (b) incubating the desired target cells in a suitable culturemedium; and (c) providing supernatent from the culture medium afterincubation with the desired target cells to dedifferentiated,programmable stem cells of human monocytic origin that are characterizedby exhibiting a CD14 antigen and a CD123 antigen to differentiate saidstem cells of human monocytic origin into target cells.

The present invention includes and provides a method of producing targetcells from dedifferentiated, programmable stem cells of human monocyticorigin comprising (a) obtaining desired target cells from a targettissue; (b) incubating the desired target cells in a suitable culturemedium; and (c) providing supernatent from the culture medium afterincubation with the desired target cells to dedifferentiated,programmable stem cells of human monocytic origin that are characterizedby exhibiting a CD14 antigen, a CD123 antigen and a CD135 antigen todifferentiate said stem cells of human monocytic origin into targetcells.

According to the present invention, the methods of producing targetcells from dedifferentiated, programmable stem cells of human monocyticorigin thus start with the isolation of desired target cells (step a),i.e. the isolation of differentiated cells of the cell type which is tobe produced using the dedifferentiated, programmable stem cells. Thedifferentiated target cells can be incubated in a cell culture medium(step b). Supernatent from the cell culture medium of the differentiatedtarget cells can be used to differentiate stem cells of human monocyticorigin into target cells (c).

The present invention includes and provides a dedifferentiated,programmable stem cell of human monocytic origin, wherein the cell ischaracterized by the membrane associated monocyte-specific surfaceantigen CD 14 and at least one pluripotency marker selected from thegroup consisting of CD117, CD123 and CD135.

The present invention includes and provides a dedifferentiated,programmable stem cell preparation comprising a dedifferentiated,programmable stem cell of human monocytic origin of the presentinvention in a suitable medium.

The present invention includes and provides a method for treating livercirrhosis using a pharmaceutical composition comprising dedifferentiatedprogrammable stem cells of the present invention.

The present invention includes and provides a method of making apharmaceutical composition for treating liver cirrhosis by preparing acomposition comprising dedifferentiated programmable stem cells of thepresent invention.

The present invention includes and provides a method for treatingpancreatic insufficiency using a pharmaceutical composition comprisingdedifferentiated programmable stem cells of the present invention.

The present invention includes and provides a method of making apharmaceutical composition for treating pancreatic insufficiency bypreparing a composition comprising dedifferentiated programmable stemcells of the present invention.

The present invention includes and provides a method for treating acuteor chronic kidney failure using a pharmaceutical composition comprisingdedifferentiated programmable stem cells of the present invention.

The present invention includes and provides a method of making apharmaceutical composition for treating acute or chronic kidney failureby preparing a composition comprising dedifferentiated programmable stemcells of the present invention.

The present invention includes and provides a method for treatinghormonal underfunctioning using a pharmaceutical composition comprisingdedifferentiated programmable stem cells of the present invention.

The present invention includes and provides a method of making apharmaceutical composition for treating hormonal underfunctioning bypreparing a composition comprising dedifferentiated programmable stemcells of the present invention.

The present invention includes and provides a method for treatingcardiac infarction using a pharmaceutical composition comprisingdedifferentiated programmable stem cells of the present invention.

The present invention includes and provides a method of making apharmaceutical composition for treating cardiac infarction by preparinga composition comprising dedifferentiated programmable stem cells of thepresent invention.

The present invention includes and provides a method for treatingpulmonary embolisms a pharmaceutical composition comprisingdedifferentiated programmable stem cells of the present invention.

The present invention includes and provides a method of making apharmaceutical composition for treating pulmonary embolisms by preparinga composition comprising dedifferentiated programmable stem cells of thepresent invention.

The present invention includes and provides a method for the treatmentof stroke using a pharmaceutical composition comprising dedifferentiatedprogrammable stem cells of the present invention.

The present invention includes and provides a method of making apharmaceutical composition for the treatment of stroke by preparing acomposition comprising dedifferentiated programmable stem cells of thepresent invention.

The present invention includes and provides a method for the treatmentof skin damage using a pharmaceutical composition comprisingdedifferentiated programmable stem cells of the present invention.

The present invention includes and provides a method of making apharmaceutical composition for the treatment of skin damage by preparinga composition comprising dedifferentiated programmable stem cells of thepresent invention.

The present invention includes and provides differentiated, isolated,somatic target cells and/or target tissue, characterized by themembrane-associated surface antigen CD14. Such cells can be obtained,for example, without limitation, by reprogramming the stem cellsaccording to a method of the present invention.

The present invention includes and provides differentiated, isolated,somatic target cells and/or target tissue characterized by themembrane-associated surface antigen CD14 where the target cells and/ortarget tissue is selected from the group consisting of adipocytes,neurons, glia cells, endothelial cells, keratinocytes, hepatocytes andislet cells.

The present invention includes and provides differentiated, isolated,somatic target cells and/or target tissue, characterized by themembrane-associated surface antigen CD14, further comprising atransfected gene. Such cells can be obtained, for example, withoutlimitation, by reprogramming the stem cells according to a method of thepresent invention.

The present invention includes and provides implantable materials coatedwith the dedifferentiated, programmable stem cells includingdifferentiated, isolated, somatic target cells and/or target tissue,obtained by reprogramming the stem cells according to a method of thepresent invention.

The present invention includes and provides implantable materials thatare prostheses, including those selected from the group consisting ofcardiac valves, vessel prostheses, bone and joint prostheses, coatedwith the dedifferentiated, programmable stem cells includingdifferentiated, isolated, somatic target cells and/or target tissue,obtained by reprogramming the stem cells according to a method of thepresent invention.

The present invention includes and provides implantable materials thatare artificial and/or biological carrier materials comprising thededifferentiated, programmable stem cells including differentiated,isolated, somatic target cells and/or target tissue, obtained byreprogramming the stem cells according to a method of the presentinvention.

The present invention includes and provides implantable materials thatare bags or chambers for introduction into the human body containingdifferentiated, isolated, somatic target cells and/or target tissue,obtained by reprogramming the stem cells according to a method of thepresent invention.

The present invention includes and provides implantable materials thatare bags or chambers, containing islet cells of the present invention,for introduction into the human body containing differentiated,isolated, somatic target cells and/or target tissue, obtained byreprogramming the stem cells according to a method of the presentinvention for the production of a pharmaceutical construct for use as anartificial islet cell portchamber for the supply of insulin.

The present invention includes and provides implantable materials thatare bags or chambers, containing adipocytes of the present invention,for introduction into the human body containing differentiated,isolated, somatic target cells and/or target tissue, obtained byreprogramming the stem cells accoding to a method of the presentinvention for the production of a pharmaceutical construct, whichcontains artificial polymers filled with adipocytes, for breastconstruction after surgery and for use in the case of plastic and/orcosmetic correction.

The present invention includes and provides implantable materials thatare semi-permeable port chamber systems comprising the dedifferentiated,programmable stem cells including differentiated, isolated, somatictarget cells and/or target tissue, obtained by reprogramming the stemcells according to a method of the present invention.

The present invention includes and provides implantable materials thatare semi-permeable port chamber systems comprising the dedifferentiated,programmable stem cells including differentiated, isolated, somatictarget cells and/or target tissue, obtained by reprogramming the stemcells according to a method of the present invention for the productionof a pharmaceutical construct for in vivo treatment of endocrine,metabolic or hemostatic diseases.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the antibody-staining of neurons and glia cells afterdifferentiation from dedifferentiated programmable stem cells. Gliacells were stained using GFAP (left-hand picture, ×200), precursor cellswere stained using S100-antigen (middle picture, ×200) and neurons usingsynaptophysin MAP2 (right-hand picture, ×200).

FIG. 2 shows endothelial cells were made visible by staining with thecorresponding endothelium-specific antibody CD31. Cells were incubatedon Matrigel for 5 days (middle picture, development of tubular strands,×200), 8 days (right picture, formation of three-dimensional networkstructures, ×200) and 12 days (left-hand picture, formation ofvessel-like three-dimensional tube, ×200).

FIG. 3 shows intermediate steps in the production of fat cells fromadult stem cells. FIG. 3A shows precursor cells containing fat vacuoles.FIGS. 3B and 3C show single adipocytes stained with Sudan red. FIG. 3Dshows aggregation and cluster formation of cells observedmacroscopically as fat tissue. FIG. 3E shows cells of monocytic origincultured in nutrient medium lacking IL-3 and 2-mercaptoethanol. FIG. 3Fshows cells treated with nutrient medium instead of FCCM after 6 days incomplete medium. FIG. 3G shows the molecular characterization, usingRT-PCR, of fat cells (adipocytes) with monocytic origin throughcomparison of the gene expression for several genes. The specificamplificates are shown by arrows indicating their size.

FIG. 4 shows development of hepatocytes from dedifferentiatedprogrammable stem cells of monocytic origin. Staining withanti-alpha-fetoprotein is shown after culture in the liver celldifferentiation medium for 6 days in FIG. 4A; for 10 days in FIG. 4B;and for 12 days in FIG. 4C. FIG. 4D shows molecular characterization,using RT-PCR, of the hepatocytes with monocytic origin throughcomparison of the gene expression for several genes. The specificamplificates are shown by arrows indicating their size.

FIG. 5 shows development of keratinocytes from the dedifferentiatedprogrammable stem cells of monocytic origin. Staining of cytokeratin 5and 6 is shown after culture in the keratinocyte differentiation mediumfor 6 days in FIG. 5A and for 10 days in FIG. 5B.

FIG. 6 shows immunohistochemical phenotyping of the cell population ofdedifferentiated programmable stem cells of monocytic origin on cytospinpreparations which had more than 70% vital cells with typical stem cellmorphology.

FIG. 7 shows in vivo differentiation of dedifferentiated programmablestem cells of monocytic origin in rats by detection of stem cells frompunch biopsies. FIG. 7A shows FISH Y-chromosome detection in stem cellderived hepatocytes after 5 days. FIG. 7B shows FISH Y-chromosomedetection in stem cells derived hepatocytes, endothelial cells, and bileduct epithelial cells after 25 days. FIG. 7C shows Kaplan-Meier survivalcurves of stem-cell treated versus untreated recipient rats followingadministration of retrorsine and 80% liver resection. FIGS. 7D and 7Eshow bilirubin and ammonia as function parameters for the completemetabolic functionality of long-term surviving stem-cell-treatedanimals.

FIG. 8 shows the insulin content of the supernatant from cultures ofinsulin-producing cells derived from programmable stem cells ofmonocytic origin measured by means of ELISA for human insulin.

FIG. 9 shows the albumin content of the supernatant from cultures ofhepatocytes derived from programmable stem cells of monocytic originmeasured by means of ELISA for human albumin.

FIG. 10 shows double-staining of the phenotypic marker for monocytes,CD14, and the liver-specific marker, albumin, to determine expression ofthe monocyte-specific antigen, CD14, and albumin in hepatocytes derivedfrom dedifferentiated stem cells.

FIG. 11 shows the results of FACS-Analysis using a FITC-marked anti-CD14antibody or a FITC-marked anti-albumin antibody to determine expressionof the monocyte-specific antigen, CD14, and the liver-specific marker,albumin, in hepatocytes derived from dedifferentiated stem cells.

DETAILED DESCRIPTION

The invention relates to adult dedifferentiated programmable stem cellsderived from human monocytes, as well as their production and use forthe production of body cells and tissues. According to a particularlypreferred embodiment of the invention these cells are autologous humanstem cells, i.e., the cell of monocytic origin comes from the patientwho is to be treated with the stem cell produced from the original celland/or with the body cells produced from this stem cell.

According to the invention this problem is solved by the production ofdedifferentiated programmable cells from human monocytes which, for thepurposes of the invention, are referred to hereafter as “stem cells”.The term “dedifferentiation” is familiar to the person skilled in therelevant art, cf. for Weissman I. L., Cell 100: 157–168, FIG. 4, (2000).It signifies the regression of an adult, already specialized(differentiated) body cell to the status of a stem cell, i.e., of acell, which in turn can be transferred (programmed) into a number ofcell types. Surprisingly, it has been demonstrated that the processaccording to the invention leads to the dedifferentiation of monocytes.The stem cells produced in this way can be transformed (programmed) intoa large number of different target cells/target tissue, cf. examples.The stem cells according to the invention express, in addition to theCD14 surface antigen characteristic of differentiated monocytes, atleast one, preferably two or three, of the typical pluripotency markersCD90, CD117, CD123 and CD135. In a particularly preferred manner, thestem cells produced according to the invention express the CD14 surfaceantigen as well as the four pluripotency markers CD90, CD117, CD123 andCD135, cf. Example 2, Table 1. Preferably, the stem cells of theinvention express the membrane associated monocyte-specific surfaceantigen CD14 and at least one pluripotency markers selected from thegroup consisting of CD117, CD123 and CD135. More preferably, the stemcells of the invention carry the CD14 antigen in combination with atleast the pluripotency marker CD123 and/or CD135. Less than 3%,preferably less than 1% of the stem cells according to the inventionexpress the CD34 antigen. Most preferably, none of the stem cells of theinvention express the CD34 antigen. In this way, for the first timeadult stem cells are made available, which can within a short time bereprogrammed into preferably autologous tissues.

The generation of the stem cells according to the invention iscompletely harmless to the patient and—in the case of autologoususe—comparable to own blood donation. The quantity of stem cells (10⁸ to10⁹ cells) required for the usual therapy options (see above) can bemade available cost-effectively within 10 to 14 days after the blood istaken. In addition the cell product provided for the therapy, in thecase of autologous use, does not give rise to any immunological problemin terms of cell rejection, as cells and recipient are preferablygenetically identical.

The stem cells according to the invention have also proved to berisk-free in animal experimentation and in culture with regard to givingrise to malignancy, a result which is only to be expected due to thecell of monocytic origin, from which the stem cells according to theinvention derive.

In one aspect, steps of the process according to the invention for theproduction of dedifferentiated programmable stem cells of humanmonocytic origin comprise:

-   -   (a) Isolation of monocytes from human blood;    -   (b) Propagating the monocytes in a suitable culture vessel        containing cell culture medium, which contains the        macrophage-colony-stimulating factor (hereafter referred to as        MCSF); and    -   (c) Cultivating the monocytes in the presence of interleukin-3        (IL-3); and    -   (d) Obtaining the human dedifferentiated programmable stem        cells, by separating the cells from the culture medium.

According to a particularly preferred embodiment of the process, M-CSFand IL-3 are simultaneously added to the cell culture medium in Step b).

It is however also possible, initially only to add M-CSF to the cellculture medium in Step b) in order to cause the monocytes to propagate,and to add IL-3 to the cell culture medium subsequently.

Finally the process in Step b) can also be carried out in such a waythat the monocytes are initially propagated in a cell culture mediumcontaining only M-CSF, then the medium is separated from the cells and asecond cell culture medium is then used, which contains IL-3.

According to a preferred embodiment of the invention the culture mediumof Step b) is separated from the cells attached to the bottom of theculture vessel and the human, dedifferentiated, programmable stem cellsare obtained by detaching the cells from the bottom and by isolating thecells.

According to a preferred embodiment of the invention the cells arefurther cultivated in the presence of a sulfur compound. The cultivationcan be carried out in a separate process step which follows thecultivation Step b) illustrated above. It can however also be carriedout in Step b), by further adding the sulfur compound to the culturemedium, preferably already at the start of the cultivation.

The process according to the invention surprisingly leads to thededifferentiation of the monocytes, wherein the adult stem cellsresulting from the dedifferentiation, besides the CD14 surface antigentypical of the differentiated monocytes, also express at least one ormore, preferably all of the pluripotency markers CD90, CD117, CD123 andCD135 (cf. Table 1). Preferably, the stem cells of the invention expressthe membrane associated monocyte-specific surface antigen CD14 and atleast one pluripotency markers selected from the group consisting ofCD117, CD123 and CD135. More preferably, the stem cells of the inventioncarry the CD 14 antigen in combination with at least the pluripotencymarker CD123 and/or CD135. Less than 3%, preferably less than 1% of thestem cells according to the invention express the CD34 antigen. Mostpreferably, none of the stem cells of the invention express the CD34antigen. The expression of the respective markers (surface antigens) canbe proved by means of commercially available antibodies with specificityagainst the respective antigens to be detected, using standard immunoassay procedures, cf. Example 2.

As the cells, during the propagation and dedifferentiation process,adhere to the bottom of the respective culture vessel, it is necessaryto separate the cells from the culture medium from Step b) and to detachthem from the bottom after completion of the dedifferentiation.According to a preferred embodiment of the invention the cell culturesupernatant is discarded before the detaching of the cells adhering tothe bottom and subsequently, the adhering cells are preferably rinsedwith fresh culture medium. Following the rinsing, fresh culture mediumis again added to the cells adhering to the bottom, and the step ofreleasing the cells from the bottom then follows (cf. Example 13).

According to a preferred embodiment the cells are brought into contactwith a biologically well-tolerated organic solvent, at the end of Stepc) and before Step d). A biologically well-tolerated organic solvent canbe an alcohol with 1–4 carbon atoms, the use of ethanol being preferred.

In a further embodiment, at the end of Step c) and before Step d) thecells are brought into contact with the vapor phase of the biologicallywell-tolerated organic solvent.

The detaching can moreover also be carried out mechanically, however, anenzymatic detaching process is preferred, for example with trypsin.

The dedifferentiated programmable stem cells obtained in this way,floating freely in the medium, can either be directly transferred to areprogramming process, or kept in the culture medium for a few days; inthe latter case, a cytokine or LIF (leukemia inhibitory factor) ispreferably added to the medium, in order to avoid premature loss of theprogrammability (cf. Donovan, P. J., Gearhart, J., loc. cit., Page 94).Finally the cells can be deep-frozen for storage purposes without lossof programmability.

The stem cells according to the invention differ from the pluripotentstem cells of embryonic origin known hitherto and from the known adultstem cells from different tissues, in that besides themembrane-associated monocyte-specific CD14 surface antigen, they carryat least one pluripotency marker from the group consisting of CD90,CD117, CD123 and CD135 on their surface. Preferably, the stem cells ofthe invention carry the membrane associated monocyte-specific surfaceantigen CD14 and at least one pluripotency markers selected from thegroup consisting of CD117, CD123 and CD135. More preferably, the stemcells of the invention carry the CD14 antigen in combination with atleast the pluripotency marker CD123 and/or CD135. Less than 3%,preferably less than 1% of the stem cells according to the inventionexpress the CD34 antigen. Most preferably, none of the stem cells of theinvention express the CD34 antigen.

The stem cells produced using the process according to the invention canbe reprogrammed into any body cells. Processes for reprogramming stemcells are known in the state of the art, cf. for example Weissman I. L.,Science 287: 1442–1446 (2000) and Insight Review Articles Nature 414:92–131 (2001), and the handbook “Methods of Tissue Engineering”, Eds.Atala, A., Lanza, R. P., Academic Press, ISBN 0-12-436636-8; Library ofCongress Catalog Card No. 200188747.

The differentiated isolated somatic target cells and/or the targettissue obtained by reprogramming of the stem cells according to theinvention moreover carry the membrane-associated CD14 differentiationmarker of the monocytes. Additionally, less than 3%, preferably lessthan 1% of these somatic target cells and/or these target tissuesaccording to the invention express the CD34 antigen. Most preferably,none of these cells or tissues express the CD34 antigen. As shown inExample 11, hepatocytes which are derived from the stem cells accordingto the invention, express the CD14 surface marker which is typical ofmonocytes, while at the same time they produce the protein albumin,which is typical of hepatocytes. The hepatocytes derived from the stemcells according to the invention can therefore be distinguished fromnatural hepatocytes. In the same way, the membrane-associated CD14surface marker was detected on insulin-producing cells, which werederived from the stem cells according to the invention (Example 9).

In one embodiment of the invention the dedifferentiated, programmablestem cells are used for the in-vitro production of target cells andtarget tissue (cf. Examples). Accordingly the invention provides methodsof producing target cells from dedifferentiated, programmable stem cellsof human monocytic origin which methods comprise the isolation ofdesired target cells as a first step (a), i.e. the isolation ofdifferentiated cells of the cell type which is to be produced using thededifferentiated, programmable stem cells. The differentiated desiredtarget cells can be in a cell culture medium (as a second step, b).Supernatent from the cell culture of the differentiated target cells canbe used to differentiate stem cells of human monocytic origin intotarget cells (in a third step, c). Illustrated methods are set forth infurther detail in Example 6 (adipocytes), Example 7 (hepatocytes) andExample 8 (keratinocytes). Therefore, differentiated, isolated tissuecells, which are obtained by differentiation (reprogramming) of the stemcells according to the invention, and which carry the membraneassociated CD14 surface antigen, are also subject of the presentinvention.

The stem cells according to the invention are preferably simply andreliably differentiated in vitro into desired target cells, such as forexample adipocytes (cf Example 6), neurons and glia cells (cf. Example3), endothelial cells (cf. Example 5), keratinocytes (cf. Example 8),hepatocytes (cf. Example 7) and islet cells (islet of Langerhans, cf.Example 9), by growing the stem cells in a medium which contains thesupernatant of the culture medium, in which the respective target cellsand/or fragments thereof have been incubated (cf. Examples 6 to 8). Thissupernatant is referred to hereafter as “target-cell-conditionedmedium”.

For the differentiation (reprogramming) of the dedifferentiated stemcells according to the invention the following procedure can thereforebe followed, in which:

-   -   a) tissue which contains or consists of the desired cells is        crushed;    -   b) the desired tissue cells and/or fragments of these are        obtained;    -   c) the desired cells and/or fragments of these are incubated in        a suitable culture medium;    -   d) the culture medium supernatant is collected during and after        the incubation as desired cell-conditioned medium; and    -   e) for the reprogramming/differentiation of dedifferentiated        stem cells into the desired cells or tissue, the stem cells are        grown in the presence of the desired-cell-conditioned medium.

Standard cell culture media can be used as culture medium (cf.Examples). The media preferably contain growth factors, such as forexample the epidermal growth factor.

The incubation of the desired cells and/or fragments of these (“cellpellet”) can be carried out over 5 to 15, preferably 10 days. Thesupernatant, i.e., the desired-cell-conditioned medium is preferablyremoved in each case after 2 to 4 days and replaced by fresh medium. Thesupernatants thus obtained can be filtered under sterile conditionsseparately or pooled and stored at approximately −20° C. or useddirectly for the programming of stem cells. As shown above, theprogramming of the stem cells into the desired target cells is carriedout by growing stem cells in the presence of the medium conditioned withthe respective desired cells (cf. Examples). The growth mediumpreferably additionally contains a desired-cell-specific growth factor,such as for example the “hepatocyte growth factor” or the “keratinocytegrowth factor” (cf. Examples).

In one embodiment of the invention the dedifferentiated, programmablestem cells according to the invention ae used per se for the productionof a pharmaceutical composition for the in-vivo production of targetcells and target tissue.

Such pharmaceutical preparations can contain the stem cells according tothe invention suspended in a physiologically well-tolerated medium.Suitable media are for example PBS (phosphate buffered saline) orphysiological saline with 20% human albumin solution and the like.

These pharmaceutical preparations contain vital dedifferentiated,programmable stem cells according to the invention, which have on theirsurface the CD14 surface marker and at least one more of the multipotentstem cell markers CD90, CD117, CD123 and/or CD135, in a quantity of atleast 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50%, preferably 60 or70%, particularly preferably 80 or 90% and extremely preferably 100%,relative to the total number of the cells present in the preparation,and optionally further pharmaceutically well-tolerated adjuvants and/orcarver substances.

Stem cell preparations can contain vital dedifferentiated, programmablestem cells according to the invention, which have on their surface theCD14 surface marker and at least one more of the pluripotent stem cellmarkers CD90, CD117, CD123 and/or CD135, in a quantity of at least 1, 2,3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58or 59%, preferably at least 60%, relative to the total number of thecells present in the preparation; cell suspensions in a cell culture- ortransport medium well-tolerated by cells, such as e.g., PBS or RPMIetc., or deep-frozen cell preparations in a suitable storage medium,such as e.g, RPMI with 50% human albumin solution and 10% DMSO arepreferred.

The number of vital cells and hence the proportion of these in thecompositions referred to above, can be determined optically by use ofthe “Trypan blue dye exclusion technique”, as vital cells can beoptically distinguished from non-vital cells, using this dye.

As a rule, it will be irrelevant for clinical use, if some of the cellspresent in the pharmaceutical preparation do not fulfil the criteria ofdedifferentiated, programmable stem cells according to the invention,provided that a sufficient number of functional stem cells is present.It is however also possible to eliminate non-dedifferentiated cells bymeans of processes known in the state of the art on the basis of surfacemarkers typical of the dedifferentiated cells according to the inventionin such preparations, so that these contain the desired cells inessentially pure form. One example of a suitable process is “Immunomagnetic bead sorting”, cf. Romani et at., J. Immunol. Methods 196:137–151 (1996).

Stem cells further have the capability, of spontaneously differentiatingin vivo by direct contact with a cell group of a specific cell type intocells of this type. Processes for tissue production using cells whichcan be redifferentiated (“tissue engineering”) are known in the state ofthe art. For example Wang, X. et al. (“Liver repopulation and correctionof metabolic liver disease by transplanted adult mouse pancreatic cells”Am. J. Pathol. 158 (2): 571–579 (2001)), have shown that even certainadult cells of the pancreas in mice are able to transform, inFAH-(fumaroylacetoacetate hydrolase)-deficient mice, into hepatocytes,which can fully compensate for the metabolic defect in these animals. Afurther example is the experiments of Lagasse et at., “Purifiedhematopoiefic stem cells can differentiate into hepatocytes in vivo”,Nature Medicine, 6 (11): 1229–1234 (2000). The authors have shown thathematopoietic stem cells from bone marrow were able, after in-vivotransfer into FAH-deficient mice, to transform into hepatocytes, whichcould then compensate for the metabolic defect; see also the review byGrompe M., “Therapeutic Liver Repopulation for the Treatment ofMetabolic Liver Diseases” Hum. Cell, 12: 171–180 (1999).

Particularly preferable forms of application for the in-vivodifferentiation of the dedifferentiated stem cells according to theinvention are injection, infusion or implantation of the stem cells intoone specific cell association in the body, in order to allow for thestem cells to differentiate there, by direct contact with the cellassociation, into cells of this cell type. For injection or infusion thecells can be administered in PBS (phosphate buffered saline).

Preferred examples of the relevant indications in this connection are:cirrhosis of the liver, pancreatic insufficiency, acute or chronickidney failure, hormonal under-functioning, cardiac infarction,pulmonary embolism, stroke and skin damage.

Therefore preferred embodiments of the invention are the use of thededifferentiated, programmable stem cells for the production ofdifferent pharmaceutical compositions for the treatment of cirrhosis ofthe liver, pancreatic insufficiency, acute or chronic kidney failure,hormonal underfunctioning, cardiac infarction, pulmonary embolism,stroke and skin damage.

For the therapeutic use of the target cells obtainable from the stemcells according to the invention, a number of concepts are available(see above Science 287: 1442–1446 (2040) and Nature 414:92–131 (2001)).

A further preferred application concerns the injection of thededifferentiated stem cells according to the invention into theperitoneum, so that they differentiate there, due to the influence ofthe cells surrounding them, into peritoneal cells. In the case ofperitoneal dialysis of patients with kidney insufficiency, these cellscan take over a kidney function via their semi-permeable membrane andgive off kidney dependent waste substances into the peritoneum fromwhere these are removed via the dialysate.

Therefore, also the differentiated, isolated, somatic target cellsand/or target tissue, which are obtained by reprogramming of the stemcells and are characterized by the membrane-associated CD14 antigen aresubject of the invention. These somatic target cells and/or targettissue preferably contain adipocytes, neurons and glia cells,endothelial cells, keratinocytes, hepatocytes and islet cells.

However the cells can also be introduced directly into the organ to bereconstituted. The introduction can be carried out via matrixconstructions which are coated with corresponding differentiated cellsor cells capable of differentiation. The matrix constructions are as arule biodegradable, so that they disappear out of the body while thenewly introduced cells grow together with the cells present. From thispoint of view, for example cellular, preferably autologous transplantsin the form of islet cells, hepatocytes, fat cells, skin cells, muscles,cardiac muscles, nerves, bones, endocrine cells etc. come underconsideration for restitution for example after partial surgicalresection of an organ, for repair for example after trauma or forsupportive use, for example in the case of lacking or insufficient organfunction.

The stem cells according to the invention and target cells obtained fromthem can further be used to coat implantable materials, in order toincrease biocompatibility. Therefore, also implantable materials, whichare coated with the dedifferentiated, programmable stem cells or thesomatic target cells and/or target tissue are subject of the invention.According to one embodiment of the invention these implantable materialsare prostheses. In particularly preferred embodiments these prosthesesare cardiac valves, vessel prostheses, bone- and joint prostheses.

The implantable materials can also be artificial and/or biologicalcarrier materials, which contain the de-differentiated, programmablestem cells or target cells. In this regards, the carrier materials canbe bags or chambers for insertion into the human body.

In one embodiment of the invention such a bag, containing islet cells,which are differentiated somatic cells according to the invention, isused for the production of a pharmaceutical construct for use as anartificial islet cell port chamber for the supply of insulin.

According to a further embodiment of the invention, a bag or chambercontaining adipocytes, which are differentiated somatic cells accordingto the invention, is used for the production of an artificial polymerfilled with adipocytes as a pharmaceutical construct for breastconstruction after surgery and in the case of further indications ofplastic and/or cosmetic correction.

Moreover, semi-permeable port chamber systems, containing endocrinecells of very widely varying provenance, can be used in vivo for thetreatment of endocrine, metabolic or hemostatic disorders. Examples ofsuch endocrine cells are cells which produce thyroxine, steroids, ADH,aldosterone, melatonin, serotonin, adrenalin, noradrenalin, TSH, LH,FSH, leptin, cholecystokinin, gastrin, insulin, glucagon, or clottingfactors.

Therefore, also implantable materials, which are semi-permeable portchamber systems, containing differentiated isolated somatic target cellsare subject of the invention. These semi-permeable chamber systems areused in different embodiments of the invention for the production of apharmaceutical construct for the in-vivo treatment of endocrine,metabolic or hemostatic disorders.

The target cells obtained from the stem cells according to the inventioncan in addition be used as cell cultures in bioreactors outside thebody, for example in order to carry out detoxification reactions. Thisform of use is particularly relevant in the case of acute conditions,for example in the case of acute liver failure as ahepatocyte-bioreactor.

The production of the constructs illustrated above and conducting thecorresponding therapeutic process have already been illustrated manytimes in the state of the art, compare for example the review by Lalan,S., et al. “Tissue engineering and its potential impact on surgery”World J. Surg. 25: 1458–1466 (2001); Nasseri, B. A., et al. “Tissueengineering: an evolving 21st century science to provide replacement forreconstruction and transplantation” Surgery 130: 781–784 (2001) andFuchs, JR., et al., “Tissue engineering: a 21 st century solution tosurgical reconstruction” AM Thorac. Surg. 72: 577–591 (2001).

Finally, the pluripotent stem cells according to the invention open up abroad field for transgenic modification and therapy. According to apreferred embodiment of the invention the dedifferentiated programmablestem cells per se or somatic target cells and/or target tissue finallydifferentiated from these, are transfected with one or more genes. Inthis way, one or more genes which are required to maintain themetabolism of certain organs, such as for example livers or kidneys, arerestored and/or supported or reintroduced. For example, stem cells orhepatocytes derived from these can be transfected with the FAH(firrnaroylacetoacetate hydrolase) gene. In the FAH deficient mousemodel the intrasplenic injection of 1000 FAH-positive donor hepatocyteswas sufficient to completely repopularize the liver after 6 to 8 weeksand fully compensate for the metabolic defect leading to cirrhosis ofthe liver (cf. Grompe, M., et al., Nat. Genet. 12: 266 ff. (1996)).

Correspondingly, by transfection of the stem cells or the respectivetarget cells obtained from the stem cells by programming (for examplehematopoietic cells, hepatocytes, ovary cells, muscle cells, nervecells, neurons, glia cells, cartilage or bones cells, etc.) with“Multi-Drug-Resistance-genes” extended radical chemotherapy can be madepossible in the case of malignant diseases by correspondinghematopoietic reconstitution or radiation resistance can be produced.

A starting material for the process according to the invention ismonocytes from human blood. These are preferably autologous monocytes,i.e., monocytes, which originate from the blood of the patient to betreated with the stem cells according to the invention or the targetcells produced from these.

To obtain the monocytes the blood can first, after standard treatmentwith an anticoagulant in a known manner, preferably by centrifugation,be separated into plasma and into white and red blood cells. After thecentrifugation the plasma is to be found in the supernatant; below thislies a layer which contains the totality of the white blood cells. Thislayer is also referred to as “buffy coat”. Below this lies the phasecontaining red blood cells (hematocrit).

The “buffy coat” layer is then isolated and separated to obtain themonocytes for example by centrifuging using a known process. Accordingto a preferred process variant the “buffy coat” layer is coated onto alymphocyte separation medium (e.g., Ficoll Hypaque) and centrifuged. Byfurther centrifuging and rinsing, the monocyte fraction is obtained fromthe blood (cf. Example 1).

Examples of alternative processes for obtaining the monocytes fromcomplete blood are “Fluorescence-Activated Cell Sorting” (FACS),“Immunomagnetic Bead Sorting” (cf. Romani et al., J. Immunol. Methods196:137–151 (1996)) and “Magnetic-Activated Cell Sorting” (MACS) or theso called “Rosetting process” (cf. Gmelig-Meyling, F., et al.,“Simplified procedure for the separation of human T and non-T cells” VoxSang. 33: 5–8 (1977)).

According to the invention, monocytes can be obtained from any isolatedhuman blood, and the blood can also originate from organs such as thespleen, lymph nodes or bone marrow. Obtaining monocytes from organs isconsidered especially when the separation of the monocytes from humanblood, e.g., in the case of anemia or leukemia, is not possible, or notin sufficient quantities, and in the case of allogenic use, if, withinthe framework of multi-organ removal, the spleen is available as asource for isolation of monocytes.

For the production of a sufficient quantity of stem cells according tothe invention it is first necessary to propagate the monocytes. For thispurpose, growth media suitable for monocytes can be used, wherein,according to the invention said medium contains M-CSF (macrophage colonystimulating factor). M-CSF (also referred to as CSF-1) is produced bymonocytes, fibroblasts and endothelial cells. The concentration of M-CSFin the culture medium can amount to 2 to 20 μg/l medium, preferably 4 to6 μg/l and in a particularly preferred manner 5 μg/l.

On the monocytes M-CSF binds to the specific c-Fms receptor (alsoreferred to as CSF-1R), which is exclusively present on the surface ofmonocytes and which only binds M-CSF (Sherr C. J., et al., Cell 41 (3):665–676 (1985)). As the specific interaction between M-CSF and thereceptor induces the division of the monocytes, the medium, in which themonocytes are cultivated contains M-CSF or an analogue thereof, whichcan bind to the receptor and activate it. Other growth factors such asGM-CSF (granulocyte-monocyte colony stimulating factor) and G-CSF(granulocyte colony stimulating factor) are unsuitable, as, due to thelack of affinity to the c-Fms receptor, they are not capable of inducingmonocyte division.

In a particularly preferred embodiment of the process M-CSF and IL-3 aresimultaneously added to the cell culture medium in Step b) of theprocess. The concentration of IL-3 in the medium may amount to 0,2 to 1μg/l, preferably 0,3 to 0,5 μg/l and in a particularly preferred manner0,4 μg IL-3/l.

It is however also possible, to add initially only M-CSF to the cellculture medium in Step b) and add IL-3 only thereafter.

In a further embodiment the culture vessel initially contains cellculture medium which contains only M-CSF, which after the separation ofthe cells is then replaced by a second cell culture medium, whichcontains IL-3.

According to a preferred embodiment of the invention the cells in Stepb) of the process are additionally cultivated in the presence of asulfur compound, e.g., a mercapto compound, in which at least onehydrocarbon group is bonded to the sulfur, and said hydrocarbon group(s)may be substituted with one or more functional groups. Mercaptocompounds are defined as compounds which have at least one mercaptogroup (—SH), which is bonded to a hydrocarbon group. By the additionaluse of such a sulfur compound, the number of the stem cells obtained bydedifferentiation of the cells of monocytic origin, which express one ormore of the stem cell markers CD90, CD117, CD123 and CD135, can beincreased.

The functional group(s) is/are preferably hydroxyl- and/or amine groups.In a particularly preferred embodiment, the sulfur compound is2-mercaptoethanol. According to a further preferred embodiment thesulfur compound is dimethylsulfoxide (DMSO).

The quantity of the sulfur compound used can range from approximately 4to approximately 200 μmol/l relative to the sulfur. Approximately 100μmol/l is preferred.

When 2-mercaptoethanol is used, the culture medium should containapproximately 3 μl to approximately 13 μl, preferably approximately 7 μl2-mercaptoethanol/l.

The treatment with IL-3 and optionally with the sulfur compound can becarried out simultaneously with or following the propagation of themonocytes by cultivation with M-CSF, simultaneous propagation andtreatment with IL-3 and optionally a sulfur compound being preferred.Propagation and dedifferentiation should, taken together, last no morethan 10 days, and the treatment with IL-3 and optionally with the sulfurcompound should be carried out over at least 3 and at most 10 days,preferably 6 days.

Therefore, according to the invention, in the case of cultivation of themonocytes in a culture medium, which simultaneously contains M-CSF, IL-3and preferably a mercapto compound, the duration of cultivation untilthe detaching of the cells from the bottom of the culture vessel amountsto at least 3 and at most 10 days, preferably 5 to 8 days andparticularly preferably 6 days.

If in a preferred embodiment the process according to the invention iscarried out in such a way that the monocytes in Step b) are initiallypropagated in a medium containing only M-CSF, the propagation in such aculture medium can take place over a period of at least 2, preferably 3and particularly preferably 4 days with a maximum duration of 7 days,and a subsequent cultivation in the presence of IL-3 and optionally of amercapto compound can take place over a further 3 days. Preferably insuch a case the cultivation in a medium containing only M-CSF willhowever only last a maximum of 4 days, followed by a cultivation in thepresence of IL-3 and optionally of a mercapto compound over a period of3, 4, 5 or 6 days.

To carry out the propagation and dedifferentiation jointly, asillustrated in Examples 2 and 13, the monocytes are after isolationtransferred into a medium, which contains both M-CSF, and IL-3 as wellas preferably the sulfur compound, in particular mercaptoethanol orDMSO.

Due to their adhesive properties the monocytes and the stem cellsproduced from them during the process adhere to the bottom of therespective culture vessel. According to a preferred embodiment of theinvention, the culture medium is after Step c) separated from the cellsadhering to the bottom of the culture vessel and is discarded. This ispreferably followed by rinsing of the cells adhering to the bottom withculture medium, and the cells are then covered with fresh culture medium(cf. Example 13).

In this step the propagation and dedifferentiation medium illustratedabove can be used as culture medium, as well as a standard cell culturemedium, for example RPMI.

According to a further preferred embodiment of the invention, the cellsare brought into contact with a biologically well-tolerated organicsolvent at the end of Step c) and before Step d), in order to increasethe number of stem cells floating freely in the medium at the end of theprocess. The quantity of the solvent can range from 10 μl to 1 ml. Thisis preferably an alcohol with 1–4 carbon atoms, the addition of ethanolbeing particularly preferred. According to a particularly preferredembodiment the cells are brought into contact with the vapor phase ofthe previously defined biologically well-tolerated organic solvent,preferably with ethanol vapor (cf. Example 2). The time for exposure tothe organic solvent, particularly preferably to ethanol vapor, shouldamount to 4–12 hours, preferably 8–10 hours.

The process according to the invention is preferably carried out inculture vessels, the surface of which has previously been coated withfetal calf serum (FCS) (cf. Example 2). Alternatively human AB-Serumfrom male donors can be also be used. The coating with FCS can be carredout by covering the surface of culture vessels with FCS before use, andafter an exposure time of a few, in particular 2 to 12 hours, and in aparticularly preferable manner 7 hours, and by removing the FCS notadhering to the surface in a suitable manner.

If treatment with organic solvent take place after Step c) optionallyafter exchange of the culture medium, the cells already become detachedfrom the bottom to a certain extent in this process step. The (further)detaching can be carried out mechanically, for example with a fine cellscraper, spatula or tip of a pipette (cf. Example 13).

According to a preferred embodiment of the process, complete detachingis carried out by treatment with a suitable enzyme, for example withtrypsin (cf. Example 2). The cells may be exposed to the trypsinsolution (0,1 to 0,025 g/l, preferably 0,05 g/l) for 2–10 minutes at 35°C. to 39° C., preferably at 37° C., in the presence of CO₂.

The trypsin activity is then blocked by a standard method, and the nowfreely floating dedifferentiated programmable stem cells can be obtainedby a standard method, for example by centrifuging and in one embodimentby suspended in a suitable cell culture at the end of Step d). They arenow available, suspended in a suitable medium, for example in RPMI 1540or DMEM, for immediate differentiation into the desired target cells.They can however also be stored in the medium for a few days. In apreferred embodiment the medium contains a cytokine or LIF factor(leukemia inhibitory factor), cf. Nature 414: 94 (2001, Donovan, P. J.,Gearhardt, J., loc. cit.), if the cells are to be stored in culture forlonger than approximately 48 hours as dedifferentiated programmable stemcells. In a medium containing such factors stem cells can be kept for atleast 10 days as dedifferentiated programmable stem cells.

In a preferred embodiment the cells are suspended for longer storage ina liquid medium and then deep-frozen. Protocols for the deep freezing ofliving cells are known in the state of the art, cf. Griffith M., et al.“Epithelial Cell Culture, Cornea, in Methods of Tissue Engineering”,Atala A., Lanza R. P., Academic Press 2002, Chapter 4, Pages 131 to 140.A preferred suspension medium for the deep freezing of the stem cellsaccording to the invention is FCS-containing DMEM, cf. Example 2.

The invention is further exemplified and illustrated below withreference to examples.

If not defined within the examples, the composition of the media andsubstances used are as follows:

1. Penicillin/streptomycin solution:

-   -   10,000 units of penicillin as sodium salt of penicillin G and        1000 μg streptomycin as streptomycin sulfate per ml        physiological sodium chloride solution (NaCI 0,9%).

2. Trypsin-EDTA

-   -   0.5 g trypsin and 0.2 g EDTA (4 Na)/l

3. Insulin

-   -   human, recombinant, produced in E. coli, approximately 28        units/mg

4. RPMI 1640 (1×, liquid (11875)) contains L-Glutamine

-   -   RPMI (Roswell Park Memorial Institute) Media 1640 are enriched        formulations, which can be used extensively for mammalian cells.

TABLE 1 Components Mol. weight Conc. (mg/l) Molarity (nM) Anorganicsalts Calcium nitrate 236 100.00 0.424 (Ca(NO₃)₂ 4H₂O) Potassiumchloride (KCl)  75 400.00 5.30 Magnesium sulfate 120 48.84 0.407 (MgSO₄)Sodium chloride (NaCl)  58 6000.00 103.44 Sodium bicarbonate  84 2000.0023.800 (NaHCO₃) Sodium phosphate 142 800.00 5.63 (Na₂HPO₄) Furthercomponents Glucose 180 2000.00 11.10 Glutathione, reduced 307 1.500.0032 Phenol red 398 5.00 0.0125 Amino acids L-Arginine 174 200.00 1.10L-Asparagine 132 50.00 0.379 L-Asparaginic acid 133 20.00 0.150L-Cysteine dihydrochloride 313 65.00 0.206 L-Glutaminic acid 147 20.000.136 L-Glutamine 146 300.00 2.05 Glycine  75 10.00 0.133 L-Histidine155 15.00 0.0967 L-Hydroxyproline 131 20.00 0.153 L-Isoleucine 131 50.000.382 L-Leucine 131 50.00 0.382 L-Lysine hydrochloride 146 40.00 0.219L-Methionine 149 15.00 0.101 L-Phenylalanine 165 15.00 0.0909 L-Proline115 20.00 0.174 L-Serine 105 30.00 0.286 L-Threonine 119 20.00 0.168L-Tryptophan 204 5.00 0.0245 L-Tyrosine disodium, 261 29.00 0.110dihydrate L-Valine 117 20.00 0.171 Vitamins Biotin 244 0.20 0.008D-calcium pantothenate 477 0.25 0.0005 Choline chloride 140 3.00 0.0214Folic acid 441 1.00 0.0022 i-Inositol 180 35.00 0.194 Niacinamide 1221.00 0.0081 p-aminobenzoic acid 137 1.00 0.0072 (PABA) Pyridoxine HCl206 1.00 0.0048 Riboflavin 376 0.20 0.0005 Thiamin HCl 337 1.00 0.0029Vitamin B12 1355  0.005 0.00000369Reference: Moore G. E., et al., J.A.M.A. 199: 519 (1967)

5. PBS (Dulbecco's phosphate buffered saline) cf. J. Exp. Med. 98:167(1954):

TABLE 2 Components g/l KCl 0.2 KH₂PO₄ 0.2 NaCl  8.00 Na₂PHO₄  1.15

6. 2-Mercaptoethanol

-   -   Quality for synthesis; Content>98%, Density 1.115 to 1.116, cf.        e.g., Momo J., et al., J. Am. Chem. Soc. 73:4961 (1951).

7. Ficoll-Hypaque:

-   -   Lymphocyte separation medium        (saccharose/epichlorohydrin-copolymerizate Mg 400,000; Density        1.077, adjusted with Sodium diatrizoate).

8. Retinic acid:

-   -   Vitamin A acid (C₂₀H₂₈O₂), 300 μl in 1.5 ml PBS corresponding to        1 mM. As medium for programming of neurons and glia cells use        150 μl on 10 ml medium (corresponding to 10⁻⁶ M).

9. DMEM

-   -   Dulbecco's modified Eagle medium (high glucose) cf. Dulbecco, R.        et al., Virology 8:396 (1959); Smith, J. D. et al., Virology        12:158 (1960); Tissue Culture Standards Committee, In Vitro 6:2        (1993)

10. L-Glutamine

-   -   Liquid: 29.2 mg/ml

11. Collagenase Type II:

-   -   Cf. Rodbell, M. et al., J. Biol. Chem. 239:375 (1964).

12. Interleukin-3 (IL-3):

-   -   Recombinant human IL-3 from E. coli (Yang Y. C. et al., Cell        47:10 (1986)); contains the 133 amino acid residues including        mature IL-3 and the 134 amino acid residues including the        methionyl form in a ratio of approximately 1:2; calculated mol.        mass approximately 17.5 kD; specific activity 1×10³ U/μg; (R&D        Catalogue No. 203-IL)

13. Macrophage-colony stimulating factor (M-CSF)

-   -   Recombinant human M-CSF from E. coli contains as monomer (18.5        kD) 135 amino acid residues including the N-terminal methionine;        is present as a homodimer with a molar mass of 37 kD; (SIGMA        Catalogue No. M 6518)

14. Antibodies:

-   -   The antibodies used in the examples against the antigens CD14,        CD31, CD90, CD117, CD123, CD135 are commercially available. They        were obtained from the following sources:    -   CD14: DAKO, Monoclonal Mouse Anti-Human CD14, Monocyte, Clone        TÜK4, Code No. M 0825, Lot 036 Edition Feb. 2, 2001;    -   CD31: PharMingen International, Monoclonal Mouse Anti-Rat CD31        (PECAM-1), Clone TLD-3A12, Catalogue No. 22711D, 0.5 mg;    -   CD90: Biozol Diagnostica, Serotec, Mouse Anti-Human CDw90, Clone        No. F15-42-1, MCAP90, Batch No. 0699;    -   CD117: DAKO, Monoclonal Mouse Anti-Human CD117, c-kit, Clone No.        104D2, Code No. M 7140, Lot 016, Edition Apr. 5, 2000;    -   CD 123: Research Diagnostics Inc., Mouse Anti-human CD123        antibodies, Clone 9F5, Catalogue No. RDI-CD123-9F5;    -   CD 135: Serotec, Mouse Anti-Human CD135, MCA1843, Clone No. BV        10A4H2.

EXAMPLE I Separation of Monocytes from Whole Blood

To avoid blood clotting and to feed the cells, 450 ml of whole blood ina 3-chamber bag set as mixed with 63 ml of a stabilizing solution, whichcontained for each liter of H₂O, 3.27 g citric acid, 26.3 g trisodiumcitrate, 25.5 g dextrose and 22.22 g sodium dihydroxyphosphate. ThepH-value of the solution amounted to 5.6–5.8.

“Sharp centrifugation” of this mixture was then carried out to separatethe blood components at 4000 rpm for 7 minutes at 20° C. This resultedin a 3-fold stratification of the corpuscular and non-corpuscularcomponents. By inserting the set of bags into a pressing machineprovided for this purpose, the erythrocytes were then pressed into thelower bag, the plasma was pressed into the upper bag, and the“Buffy-coat” remained in the middle bag, and it contained approximately50 ml in volume.

The quantity of 50 ml freshly obtained “Buffy-coat” was then dividedinto 2 portions of 25 ml each, each of which was then coated with 25 mlFicoll-Hypaque separation medium, which had been introduced into two 50ml Falcon tubes beforehand.

This mixture was centrifuged without brake for 30 minutes at 2500 rpm.Thereafter, erythrocytes and dead cells still present in the “Bullycoat” lay below the Ficoll phase while the white blood cells includingthe monocytes are separated as a white interphase on the Ficoll.

The white interphase of the monocytes was then carefully pipetted offand was mixed with 10 ml of phosphate buffered physiological saline(PBS).

This mixture was then centrifuged with brake three times for 10 minutesat 1800 rpm; the supernatant was pipetted off after each centrifugationand fresh PBS was filled up.

The cell sediment collected on the base of the centrifugation vessel(Falcon tube) contained the mononuclear cell fraction, i.e., themonocytes.

EXAMPLE 2 Propagation and Dedifferentiation of the Monocytes

The cultivation and propagation of the monocytes on the one hand and thededifferentiation of the cells on the other hand were carried out in onestep in nutrient medium of the following composition:

TABLE 3 RPMI 1640 medium 440 ml Fetal calf serum (FCS)  50 mlPenicillin/Streptomycin solution  5 ml 2-Mercaptoethanol (Stocksolution)  5 ml Total volume 500 ml

The nutrient medium further contained 2.5 μg 1500 ml of M-CSF and 0.2 μg1500 ml interleukin-3 (IL-3).

The monocytes isolated in Example 1 were transferred into 5 chambers ofa 6-chamber well plate (30 mm diameter per well) in a quantity ofapproximately 10⁵ cells per chamber in each case, and filled up in eachcase with 2 ml of the above-mentioned nutrient medium. The 6-well platewas previously filled with pure, inactivated FCS and the FCS wasdecanted after approximately 7 hours, in order to obtain an FCS-coatedplate in this way. The cell number for the exact dose per well wasdetermined according to a known process, cf. Hay R. J., “CellQuantification and Characterization” in Methods of Tissue Engineering,Academic Press 2002, Chapter 4, Pages 55–84.

The 6-well plate was covered with its lid and stored for 6 days in anincubator at 37° C. The cells settled to the bottom of the chambersafter 24 hours. Every second day the supernatant was pipetted off andthe chambers of the 6-well plate were again each filled up with 2 ml offresh nutrient medium.

On the 6th day 2 ml of 70% ethanol was introduced into the 6-wellplate's 6th chamber which had remained free, the plate was again closedand was stored for a further 10 hours at 37° C. in the incubator.

Subsequently, 1 ml of a trypsin solution diluted 1:10 with PBS werepipetted into each of the chambers of the well plate which containedcells. The closed well plate was placed for 5 minutes at 37° C. under 5%CO₂ in the incubator.

The trypsin activity was subsequently blocked by the addition of 2 ml ofRPMI 1640 medium to each of the wells. The total supernatant in each ofthe chambers (1 ml trypsin+2 ml medium) was pipetted off, pooled in a 15ml Falcon tube and centrifuged for 10 minutes at 1800 rpm. Thesupernatant was then discarded and the precipitate was mixed with freshRPMI 1640 medium (2 ml/10⁵ cells).

This cell suspension could be directly used for differentiation intodifferent target cells.

Alternatively, after centrifugation and discarding of thetrypsin-containing supernatant the cells were mixed with DMSO/FCS as afreezing medium and deep-frozen at a concentration of 10⁶/ml.

The freezing medium contained 95% FCS and 5% DMSO. In each caseapproximately 10⁶ cells were taken up in 1 ml of the medium and cooleddown in the following steps:

-   -   30 minutes on ice;    -   2 hours at −20° C. in pre-cooled Styropor boxes;    -   24 hours at −80° C. in Styropor;    -   Storage in tubes in liquid nitrogen (N₂) at −180° C.

For immune-histochemical phenotyping of the cell population ofdedifferentiated programmable stem cells of monocytic origin, generatedaccording to the above process, in each case 10⁵ cells were taken andfixed as a cytospin preparation on slides for further histochemicalstaining (Watson, P. “A slide centrifuge; an apparatus for concentratingcells in suspension on a microscope slide.” J. Lab. Clin. Med., 68:494–501 (1966)). After this the cells could be stained using thetechnique illustrated by Cordell, J. L., et al., (Literature, see below)with APAAP red complex. If not indicated otherwise, the added primaryantibody was diluted 1:100 with PBS, and in each case 200 μl of thisconcentration of antibodies was used. Monoclonal antibodies were used asprimary antibodies against the cell antigen epitopes listed in Table 1.FIG. 6 shows stained cytospin preparations and the corresponding proofof the stem cell markers CD90, CD117, CD123 and CD135.

Literature Relating to Staining Technique:

Cordell J. L., et al. “Immunoenzyrnatic labeling of monoclonalantibodies using immune complexes of alkaline phosphatase and monoclonalanti-alkaline phosphatase (APAAP complexes).” J. Histochem. Cytochem.32: 219–229 (1984).

Literature Relating to the Markers:

CD14

-   Ferrero E., Goyert S. M. “Nucleotide sequence of the gene encoding    the monocyte differentiation antigen, CD14”, Nucleic Acids Res.    16:4173–4173 (1988).    CD31-   Newman P. J., Berndt M. C., Gorski J., White J. C. H, Lyman S.,    Paddock C., Muller W. A. PECAM-1 (CD31) cloning and relation to    adhesion molecules of the immunoglobulin gene superfamily”, Science    247:1219–1222 (1990).    CD90-   Seki T., Spur N., Obata F., Goyert S., Goodfellow P., Silver J. “The    human thy-1 gene: structure and chromosomal location”, Proc. Natl.    Acad. Sci. USA 82:6657–6661 (1985).    CD117-   Yarden Y., Kuang W. J., Yang-Feng T., Coussels L., Munemitsu S.,    Dull T. J., Chen E., Schlessinger J., Francke U., Ullrich A. “Human    proto-oncogene c-kit: a new cell surface receptor tyrosine kinase    for an unidentified ligand.” EMBO J. 16:3341–3351 (1987).    CD123-   Kitamura T., Sato N., Arai K., Miyajima A. “expression cloning of    the human IL-3 receptor cDNA reveals a shared beta subunit for the    human IL-3 and GM-CSF receptors.” Cell 66:165–1174 (1991).    CD135-   Small D., Levenstein M., Kim E., Carow C., Amn S., Rockwell P.,    Witte L., Burrow C., Ratajazak M. Z., Gewirtz A. M., Civin C. I.,    “STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed    in CD34+ human bone marrow cells and is involved in the    proliferation of early progenitor/stem cells.” Proc. Natl. Acad.    Sci. USA 91:459–463 (1994).

TABLE 4 Antigen expression of the stem cells according to the inventionAntigen Color reaction Stem cell marker CD90 ++ CD117 + CD123 ++ CD135+(+) Differentiation marker CD14 (monocytes) + + clear color reaction ofthe cells with the primary antibody; ++ strong color reaction of thecells with the primary antibody.

The graduation indicated corresponds to the detected antigen positivity,which becomes apparent from Day 4 to Day 9 after cultivation of themonocytes in the correspondingly specified media and was carried out viamicroscopic comparison of the respective cytospin colorations with thenegative control (coloration observed without primary antibodies).

Only cytospin preparations which had more than 70% vital cells withtypical stem cell morphology (cf. FIG. 6) were evaluated. Less than 1%of these cells expressed the CD34 antigen.

EXAMPLE 3 Production of Neurons and Glia Cells from Adult Stem Cells

The production of neurons and glia cells was carried out in petri disheswith a diameter of 100 mm. To prepare the petri dishes, 5 ml of pureinactivated fetal calf serum (FCS) was introduced into each dish, sothat the bottom was covered. After 7 hours, the proportion of FCS notadhering to the bottom of the petri dish was pipetted off. Approximately10⁶ of the cells produced in accordance with Example 2 were introducedinto one of the prepared petri dishes and 10 ml of nutrient medium ofthe following composition was added:

TABLE 5 DMEM solution 440 ml Fetal calf serum (FCS)  50 ml 1-Glutamine 5 ml Penicillin (100 U/l)/Streptomycin (100 μg/l) solution  5 ml Totalvolume 500 ml

The nutrient medium further contained retinic acid in a quantity of1×10⁶ M/500 ml.

The reprogramming/differentiation of the stem cells used into neuronsand glia cells took place within 10 days, the medium being changed atintervals of approximately 3 days. After this period, the cells weremostly adhering to the bottom of the chamber and could be detached bybrief trypsinization from the bottom of the plate in a manner analogousto that previously illustrated for the stem cells.

EXAMPLE 4 Evidence of Neuronal Precursor Cells, Neurons and Glia Cells

For the later immunohistochemical characterization of the target cellsinduced by the dedifferentiated programmable stem cells, the stem cellsgenerated from monocytes (10⁵ cells/glass lid) were applied to glasslids (20 mm×20 mm), which were placed on the bottom of the 6-well plates(30 mm diameter per chamber) and cultivated with the nutrient medium (2ml) per well plate. After the respective target cells weredifferentiated, these were fixed as follows: After removal of thenutrient medium (supernatant) the cultivated target cells were fixed bythe addition of 2 ml Methanol, which took effect over 10 minutes.Subsequently the ethanol was pipetted off, and the well plates werewashed twice with PBS (2 ml in each case). After this, the cells couldbe stained with APAAP red complex using the technique illustrated byCordell, J. L., et al., “Immunoenzymatic labeling monoclonal antibodiesusing immune complexes of alkaline phosphatase and monoclonalanti-alkaline phosphatase (APAAP complexes).” J. Histochem. Cytochem.32:219–229 (1994). Unless otherwise specified, the added primaryantibody was diluted 1:100 with PBS, in each case 200 μl of thisconcentration of antibodies were pipetted into each of the 6 wells.

Neuronal precursor cells were detected by staining the cells with theantibody against the S100-antigen, cf. middle picture of FIG. 1 (×200).

Neurons were detected by specific expression of synaptophysin MAP2(microtubular associated protein 2) or neurofilament 68 with thecorresponding specific antibodies (primary antibody diluted 1:300 withPBS), right-hand picture of FIG. 1, ×200.

Glia cells, such as for example astrocytes, were identified by detectionof GFAP (glial fibrillary associated protein) (primary antibody diluted1:200 with PBS), left-hand picture of FIG. 1, ×200.

The separation of neurons and glia cells was carried out usingantibodies specific against MAP2 (neurons) or GFAP (glia cells), bymeans of MACS (Magnetic Activated Cell Sorting) according to the processas illustrated for example in Carmiol S., “Cell Isolation and Selection”Methods of Tissue Engineering, Academic Press 2002, Chapter 2, Pages19–35.

The cell types made visible by staining are shown in FIG. 1.

EXAMPLE 5 Production of Endothelial Cells from DedifferentiatedProgrammable Adult Stem Cells of Monocytic Origin

For the cultivation of endothelial cells, Matrigel® (Beckton andDickinson, Heidelberg, Del.) was used as matrix. This matrix consists offibronectin, laminin and collagen I and IV.

The frozen matrix was slowly thawed at 4° C. in a refrigerator over aperiod of 12 hours. During this period its state changed, i.e., theoriginally solid matrix became spongy/liquid. In this state it wasintroduced into a 48-well plate (10 mm diameter per well) in such amanner, that the bottom of each of the wells was covered.

After application, the plate was kept for 30 minutes at roomtemperature, until the gell had solidified at the bottom as an aherentlayer.

Subsequently approximately 1×102 cells per well were incubated onMatrigel® with addition of the nutrient medium (as illustrated inExample 2).

After 4–5 days the first tubular cell strands appeared, which developedafter 6–8 days into three-dimensional cell networks. On the cells, theendothelial markers CD31 and factor VIII could be identified with therespective specific primary antibodies (200 μl, in each case diluted to1:100 with PBS).

In an alternative process the liquefied matrix was applied to avessel-prosthesis, which was then coated with the dedifferentiatedprogrammable adult stem cells according to Example 2. Afterapproximately 6 days a lawn of endothelial cells could be identified,which coated the prosthesis in a circular manner.

The endothelial cells made visible by staining with correspondingendothelium-specific antibodies (see above) are shown in FIG. 2. In themiddle picture, the cells are shown after 5 days' incubation onMatrigel®. First tubular strands combine individual cell aggregates. Thedark-brown marked cells express CD31 antigen (×200 with yellow filter).After 8 days there is an increasing formation of three-dimensionalnetwork structures takes place (anti-CD31-antigen staining, ×200 withyellow filter). After 12 days the newly differentiated CD31⁺ cells,which had been cultivated on Matrigel®, form a vessel-likethree-dimensional tube with multi-layer wall structures, which isalready morphologically reminiscent of a vessel. It is recognized, thatnow almost all the cells express the CD31 antigen (CD31 coloration,×200, blue filter), left-hand picture.

EXAMPLE 6 Production of Fat Cells (Adipocytes)

A. For the programming/differentiation of the adult stem cells accordingto Example 2 into fat cells, a conditioned medium was first generated.For this purpose 20 g of an autologous fat tissue, i.e., fat tissue fromthe same human donor, from the blood of whom the monocytes alsooriginated, was processed as follows:

At first, the fat tissue was crushed in a petri dish and the crushedtissue pieces were passed through a sieve (diameter of holes 100 μm).

The suspension thus obtained was then transferred into a petri dish witha diameter of 100 mm and 10 ml DMEM-medium with a content of 30 mgcollagenase type II were added. The mixture was left for approximately60 minutes at room temperature (22° C.−±2° C.) to allow the collagenaseto take effect on the fat cells.

Subsequently the mixture was transferred to 50-ml Falcon tubes, and thetubes were centrifuged for 10 minutes at 1800 rpm.

After centrifugation the supernatant was discarded and the cell pelletconsisting of adipocytes and precursor cells was taken up in 8 ml of amedium of the following composition and incubated in petri dishes(diameter 100 mm) for 10 days at 37° C. in an incubator:

TABLE 6 DMEM solution 444.5 ml Fetal calf serum (FCS)   50 ml Insulinsolution  0.5 ml Penicillin (100 U/l)/Streptomycin (100 μg/l) solution   5 ml Total volume   500 ml

The insulin solution contained 18 mg insulin (Sigma 1-0259) dissolved in2 ml of acetic water (consisting of 40 ml of H₂O and 0.4 ml of glacialacetic acid). The solution is diluted 1:10 with acetic water.

During the incubation over 10 days, the fat-cell-conditioned medium(FCCM) formed a supernatant. The supernatant was replaced with freshnutrient medium after 2 to 4 days in each case. The FCCM obtained duringeach change of medium was subjected to sterile filtration and stored at−20° C. Subsequently 10 ml of the FCCM illustrated above were introducedinto a petri dish (diameter 100 mm) together with approximately 10⁶ stemcells according to Example 2. The first precursor cells containing fatvacuoles became visible after 4 days (FIG. 3A). After 6 days, singleadipocytes appeared, which could be stained with Sudan red (FIGS. 3B andC). After 10 days there was typical aggregation and cluster formation ofthese cells, which at this step could already be observedmacroscopically as fat tissue (FIG. 3D).

The fat cells made visible by staining in FIGS. 3A–3D thus differ quiteconsiderably from the controls 3E and 3F: FIG. 3E shows the cells ofmonocytic origin, which were cultivated in the nutrient medium (asindicated in Example 2) for 6 days, but without the addition of IL-3 and2-mercaptoethanol to the nutrient medium. This was followed by theaddition of the FCCM. These cells were not capable of differentiatinginto fat cells. Figure F shows cells, which were cultivated for 6 dayswith complete medium (according to Example 2), and which were thentreated for a further 6 days with nutrient medium instead of with FCCM(according to Example 2). The FCCM thus contains components which arerequired to provide the signal for differentiation into fat cells.

The staining of the cells with Sudan red in FIGS. 3A, B, C and D tookplace according to the method illustrated by Patrick Jr., C. W., et al.“Epithelial Cell Culture: Breast”, in Methods of Tissue Engineering,Academic Press 2002, Chapter 4, Pages 141–149.

B. In addition to the phenotyping of the fat cells by staining withSudan red, molecular-biological characterization of the fat cells wascarried out at the mRNA level, in order to check whether the geneticprogram of the fat cells, after corresponding programming with thefat-cell-conditioning medium used, undergoes a corresponding alteration,and typical messenger-ribonucleic acid (mRNA) transcripts, illustratedfor fat cells can be identified in the fat cells programmed fromprogrammable monocytes. Two mRNA sequences typical of fat cellmetabolism were amplified by means of polymerase chain reaction (PCR)from isolated RNA samples from dedifferentiated programmable stem cellsof monocytic origin and, in a parallel test mixture, amplified from theprogrammed fat cells, namely “peroxisome proliferative activatedreceptor gamma” (PPARG)-mRNA, (Tontonoz, P., et al. “Stimulation ofadipogenesis in fibroblasts by PPAR gamma 2, a lipid-activatedtranscription factor.” Cell 79: 1147–1156 (1994), gene bank access codenumber; NM_(—)005037) and “leptin (obesity homolog, mouse)”-mRNA, (ZhangY., et al. “Positional cloning of the mouse obese gene and its humanhomologue.” Nature 372: 425–432 (1994), gene bank, access code number:NM_(—)000320).

The RNA-isolation needed for this purpose, the reverse transcriptionmethod and the conditions of the PCR amplification of the desired mRNAsequences were carried out as illustrated in detail in the state of theart, see Ungefroren H., et al., “Human pancreatic adenocarcinomasexpress Fas and Fas ligand yet are resistant to Fas-mediated apoptosis”,Cancer Res. 58: 1741–1749 (1998).

For this purpose the respective primers produced for the PCRamplification were selected so that the forward- and reverse primersbind to mRNA sequences, whose homologous regions in the chromosomal genelie in two different exons and are separated from one another by a largeintron. It could thereby be ensured that the amplification fragmentobtained originates from the mRNA contained in the cell and not from thesequence present in the chromosomal DNA. In particular the followingprimer sequences were selected for PPAR-γ and for leptin:

PPAR-γ: forward-primer; 265–288 (corresponding gene sequence in exon 1),reverse-primer: 487–465 (corresponding gene sequence in exon 2), thisresults in an amplification fragment of 487–265 by =223 bp, see FIG. 3G.As further shown by FIG. 3G traces of transcribed PPARγ-specific mRNAcan already be identified in the programmable stem cell and in the tumorcell line HL-60 (of a human promyeloic leukemia cell line), althoughwith significantly narrower signal bands than in the fat cell itself. Incontrast, the fat-cell-specific protein leptin can only be detected inthe fat cells derived from the programmable stem cells at mRNA level byreverse-transcriptase PCR.

The programmable stem cells (progr. stem cell) used as a control and thehuman tumor cell lines HL-60, Panc-1 and WI-38 transcribe no leptin. Asnegative controls all the samples without the addition of the reversetranscriptase (fat cell/-RT) and H₂O-samples were simultaneouslyco-determined. By identification of the GAPDH “house-keeping” gene inthe positive controls, it is ensured that the respective PCRamplification steps were properly carried out in the individualmixtures.

EXAMPLE 7 Production of Liver Cells (Hepatocytes)

A. For the programming of the dedifferentiated programmable stem cellsof monocytic origin according to Example 2 into liver cells, aconditioned medium was first generated. For this purpose 40 g of humanliver tissue was processed as follows.

First the liver tissue was rinsed several times in PBS, to essentiallyremove erythrocytes. The tissue was then crushed in a petri dish andincubated with a dissociation solution for approximately 45 minutes atroom temperature. The dissociation solution consisted of 40 ml PBS(phosphate buffered saline), 10 ml of a trypsin solution diluted 1:10with PBS and 30 mg collagenase type H (Rodbel M., et al. J. Biol. Chem.239: 375 (1964)). After a 45-minutes incubation, the tissue pieces werepassed through a sieve (see Example 6).

The mixture was then transferred into 50-m1 Falcon tubes, filled up to50 ml with PBS and centrifuged for 10 minutes at 1800 rpm.

After centrifugation the supernatant was discarded and the cell pelletcontaining the liver cells was again washed with 50 ml PBS andcentrifuged. The supernatant thus produced was again discarded and thecell pellet taken up in 25 ml of a medium of the following compositionand incubated in cell culture flasks (250 ml volume) for 10 days at 37°C. in an incubator:

TABLE 7 Liver cell growth medium Liver cell growth medium, LCGM RPMI1640 medium 445 ml Fetal calf serum (FCS)  50 ml Insulin solution  0.5ml  Penicillin (100 U/l)/Streptomycin (100 μg/l) solution  5 ml Totalvolume 500 ml

The nutrient medium contained in addition 5 μg (10 ng/ml) of epidermalgrowth factor (Pascall, I. C. et al., J. Mot. Endocrinol. 12: 313(1994)). The composition of the Insulin solution was as illustrated inExample 6.

During the incubation lasting 10 days the liver cell conditioned medium(LCCM) formed as a supernatant. The supernatant was replaced by freshnutrient medium after 2 to 4 days respectively. The respective LCCMobtained during the change of medium in each case was subjected tosterile filtration (filter with 0.2 μm pore size) and stored at −20° C.,

1×10⁶ dedifferentiated stem cells were then cultivated with 10 ml of amedium of the following composition in a petri dish (Ø100 mm) or aculture flask.

TABLE 8 Liver cell differentiation medium (Liver cell differentiationmedium, LCDM): LCCM 100 ml Insulin solution (cf. Example 6) 0.1 mlepidermal growth factor 1 μg hepatocyte growth factor 2 μg

Hepatocyte growth factor (Kobayashi, Y. et al., Biochem. Biophys. Res.Commun. 220: 7 (1996)) was used in the concentration of 40 ng/ml. Aftera few days morphological changes towards flat, polygonal mono- ordiploid cells could be observed (FIG. 4A). After 10–12 days hepatocytesarising from dedifferentiated stem cells could be identified byimmune-histochemical detection of the liver-specific antigenalpha-fetoprotein (Jacobsen, G. K. et al., Am. J. Surg. Pathol. 5:257–66 (1981)), as shown in FIGS. 4B and 4C.

B. In addition to the phenotyping of the hepatocytes byimmune-histochemical identification of the alpha-fetoprotein, amolecular-biological characterization of the hepatocytes at mRNA levelwas carried out, in order to check whether the genetic program of thestem cells, after corresponding programming with theliver-cell-conditioning medium used undergoes a correspondingalteration, and whether messenger-ribonucleic acid (mRNA) transcripts,illustrated as typical of liver cells in the hepatocytes arising fromthe stem cells according to the invention can be identified. For thispurpose, the presence of five different mRNA sequences typical ofhepatocytes was examined by means of polymerase chain reaction (PCR) inisolated RNA samples from dedifferentiated programmable stem cells ofmonocytic origin and, in a parallel test sample, from the liver cellsobtained by programming of the stem cells. In particular, this is theHomo sapiens albumin-mRNA (Lawn, R. M., et al. “The sequence of humanserum albumin cDNA and its expression in E. coli.” Nucleic Acids Res. 9:6103–6114, (1981), gene bank access code number: NM-000477),alpha-fetoprotein-mRNA (Morinaga T., et al. “Primary structures of humanalpha-fetoprotein and its mRNA.” Proc. Natl. Acad. Sci. USA 80:4604–4608 (1983), gene bank access code number: V01514), Human carbamylphosphate synthetase I mRNA (Haraguchi, Y., et al. “Cloning and sequenceof a cDNA encoding human carbamyl phosphate synthetase I: molecularanalysis of hyper-ammonemia” Gene 107: 335–340 (1991), gene bank accesscode number D90282), Homo sapiens coagulation factor 11 (Thrombin, F2)mRNA (Degen, S. J. et al. “Characterization of the complementarydeoxyribonucleic acid and gene coding for human prothrombin”Biochemistry 22: 2087–2097 (1983), gene bank access code numberNM-000506), Homo sapiens coagulation factor VII (serum prothrombinconversion accelerator, F7) mRNA (NCBI Annotation Project. DirectSubmission, 06 Feb. 2002, National Center for Biotechnology Information,NIH, Bethesda, Md. 20894, USA, gene bank access code number XM-027508),

The RNA-isolation necessary for this reverse transcriptase method andthe conditions of the PCR amplification of the desired mRNA sequenceswas carried out as illustrated in detail in the state of the art, seeUngefroren H., et al., “Human pancreatic adenocarcinomas express Fas andFas ligand yet are resistant to Fas-mediated apoptosis” Cancer Res. 58:1741–1749 (1998).

The respective primers for the PCR amplification were selected so thatthe forward- and reverse primers bind to mRNA sequences whose homologousregions in the chromosomal gene lie in two different exons and areseparated from one another by a large intron. In this way it could beensured that the amplification fragment obtained originates from themRNA contained in the cell and not from the sequence present in thechromosomal DNA.

The primer sequences indicated below were selected; the results of therespective PCR analyses are reproduced in FIG. 4D. The dedifferentiatedprogrammable stem cells according to the invention, are designated thereas “progr. stem cell” and the hepatocytes derived by programming ofthese as “progr. hepatocyte”.

Alpha-fetoprotein: forward primer: 1458–1478 (corresponding genesequence in Exon 1), reverse primer: 1758–1735 (corresponding genesequence in Exon 2), this results in an amplification fragment of1758–1458 by =391 bp, see FIG. 4D.

As shown in FIG. 4, the programmable stem cell (progr. stem cell), whichitself contains no identifiable specific mRNA transcripts foralpha-fetoprotein, can be programmed into a hepatocyte (progr.hepatocyte), which contains this mRNA transcript (positive band with amolecular weight of 301 bp). This also explains the immune-histochemicaldetectability of the alpha-fetoprotein, as shown in FIGS. 4B and 4C. Thepositive controls, namely human liver tissue and the liver tumor cellline HepG2 also transcribe alpha-fetoprotein-specific mRNA, as the 301bp bands confirm.

Albumin: forward primer: 1450–1473 (corresponding gene sequence in exon1), reverse primer: 868–1844 (corresponding gene sequence in Exon 2),this resulted in an amplification fragment of 1868–1450 by =419 bp, seeFIG. 4D.

FIG. 4D shows traces of transcribed albumin-specific MRNA already in theprogrammable stem cell, while the hepatocytes obtained by programming ofthe stem cells and normal liver tissue as well as the tumor cell lineHepG2, which were both used as positive controls, strongly express themRNA, as can be recognized by clear bands.

The carbamyl phosphatase synthetase I: forward primer: 3135–3157(corresponding gene sequence in exon 1), reverse primer. 4635–4613(corresponding gene sequence in exon 2), this results in anamplification fragment of 4635−3135=1500 bp, see FIG. 4D.

The carbamyl phosphate synthetase I represents an enzyme specific to thehepatocytes, which plays an important role in the metabolization of ureain the “urea cycle”. This detoxification function is guaranteed byfunctioning hepatocytes. As FIG. 4D shows, both in the hepatocytesgenerated from programmable stem cells and also in the positive controls(human liver tissue and the HepG2-tumor cell line), the mRNA bands (1500bp) specific to carbamyl phosphate synthetase I can be identified. Thesomewhat weaker expression of the mRNA bands for the programmedhepatocytes (progr. hepatocyte) is due to the lack of substrateavailable in the culture dish.

Clotting factor II: forward primer: 1458–1481 (corresponding genesequence in exon 1), reverse primer: 1901–1877 (corresponding genesequence in exon 2), this results in an amplification fragment of1901−1458=444 bp, see FIG. 4D.

This likewise hepatocyte-specific protein can only be detected in theprogrammed hepatocyte (progr. hepatocyte) and in the positive controlfrom human liver tissue at mRNA level by 444 by band expression, whereasthe programmable stem cell (progr. stem cell) does not show this band,i.e., the gene is not transcribed there, as can be seen in FIG. 4D.

Clotting factor VII: forward primer: 725–747 (corresponding genesequence in exon 1), reverse primer: 1289–1268 (corresponding genesequence in exon 2), this results in an amplification fragment of1289−725=565 bp, see FIG. 4D.

As in the case of clotting factor II, also this protein is onlytranscribed in programmed hepatocytes (progr. hepatocyte) and in thepositive control (human liver tissue) (see bands at 656 bp), althoughweaker than clotting factor II. Neither the programmable stem cell northe negative control (H₂O) show this specific mRNA band.

Glycerine aldehyde dehydrogenase: This gene, also referred to as a“house-keeping gene” can be detected in every eukaryotic cell and servesas a control whether PCR amplification was properly carried out in allsamples; it is co-determined in parallel and results from the additionof a definite quantity of RNA from the respective cell samples.As negative control H₂O samples were simultaneously co-determined in alltests. If the H₂O is not contaminated with RNA, no amplificate isproduced during the PCR and no band is detectable (thus serves ascounter-control).

EXAMPLE 8 Production of Skin Cells (Keratinocytes)

For the programming of dedifferentiated programmable stem cells ofmonocytic origin according to Example 2 in skin cells a conditionedmedium was first generated. For this purpose, 1–2 cm² of complete humanskin was processed as follows.

The skin material was first freed from the subcutis under sterileconditions. The tissue was then washed a total of ten times with PBS ina sterile container by vigorous shaking. After the second washing, thetissue was again freed from demarked connective tissue residues.

The skin material was then placed in a petri dish with a diameter of 60mm, mixed with 3 ml of a trypsin solution diluted 1:10 with PBS and cutinto small pieces (approximately 0.5 to 1 mm³). After this, 3 ml of thetrypsin solution diluted 1:100 with PBS was again added to the mixtureand the mixture was incubated at 37° C. for 60 minutes with intermittentshaking.

The larger particles were then allowed to settle and the supernatantcontaining the keratinocytes was poured off and centrifuged at 800 rpmfor 5 minutes. The supernatant now produced was pipetted off and thecell pellet was taken up in 3 ml of a medium of the followingcomposition and incubated in petri dishes (Ø100 mm) for 15 days in anincubator at 37° C.

TABLE 9 Keratinocyte growth medium (Keratinocyte growth medium, KGM):DMEM 333.5 ml Fetal Calf serum (FCS)   50 ml Ham's F12 medium   111 mlPenicillin (100 U/l)/Streptomycin (100 μg/l) solution    5 ml Insulinsolution (cf. Example 6)  0.5 ml Total volume   500 ml

The nutrient medium contained 5 μg of epidermal growth factor (for exactspecification see Example 7) and 5 mg of hydrocortisone (Ref. MerckIndex: 12, 4828).

During the 15 days' incubation period, the keratinocyte-cell-conditionedmedium KCCM formed as supernatant. The supernatant was replaced withfresh nutrient medium after 2–4 days in each case. The KCCM obtainedduring each change of medium was subjected to sterile filtration andstored at −20° C.

1×10⁶ dedifferentiated stem cells were then cultivated with 10 ml of amediums of the following composition in a petri dish (Ø100 mm) or aculture flask.

TABLE 10 Keratinocyte differentiation medium (Keratinocytedifferentiation medium, KDM) KCCM 100 ml Insulin solution (cf. Example6) 0.5 ml epidermal growth factor (EGF) 1 μg Hydrocortisone 1 mgkeratinocyte growth factor (KGF) 2.5 μg

Keratinocyte growth factor was used in a concentration of 25 ng/ml, asillustrated by Finch et al., Gastroenterology 110: 441 (1996).

After a few days a morphological change in the cells could be observed.After 6 days the keratinocyte-specific antigens, cytokeratin 5 and 6,which are both bound by the primary antibody used, (Exp. Cell. Res. 162:114 (1986)) could be detected (FIG. 5A). After 10 days a cell adherenceof the clearly larger individual cells already took place in culture,which made it possible to identify a visible cell tissue combination ofconfluent cells (FIG. 5B).

EXAMPLE 9 Production of Insulin-Producing Cells from DifferentiatedProgrammed Stem Cells

The production of insulin-producing cells was conducted in cultureflasks with a volume of approximately 250 ml and flat walls (T75 cellculture flasks). Approximately 5×10⁶ of the cells produced according toExample 13 were suspended in approximately 5 ml of the culture mediumindicated below (differentiation medium for insulin producing cells) andafter being introduced into the flasks, mixed with a further 15 ml ofculture medium. For the differentiation of the cells, the flasks wereincubated in a horizontal position in an incubator at 37° C. and 5% CO₂.

Culture medium (modified according to Rameya V. K. et al., NatureMedicine, 6 (3), 278–282 (2000)):

TABLE 11 RPMI 1640 445 ml Fetal calf serum (FCS) 50 ml Penicillin (100U/l)/Streptomycin (100 μg/l) solution 5 ml Nicotinamide 620 mg Glucose360 mg Total volume 500 ml

The nutrient medium further contained the epidermal growth factor in aquantity of 10 ng/ml and the hepatocyte growth factor in a quantity of20 ng/ml.

Within the first hour the cells adhere to the bottom of the culturevessel. The differentiation of the stem cells was monitored by referenceto insulin production. For this purpose the culture medium was changedat intervals of approximately 2 to 3 days, the cell supernatant wascollected each time, and frozen at −20° C. The cells adhering to thebottom of the culture flask could be detached by tryptinization asillustrated in Example 2.

The insulin content of the supernatant collected at the different timeswas measured by means of ELISA (Enzyme-linked-immunosorbent-assay)against human insulin (Bruhn H. D., Fölsch U. R. (Eds.), Lehrbuch derLabormedizin: Grundlagen, Diagnostik, Klinik Pathobiochemie [Textbook ofLaboratory Medicine, Principles, Diagnosis, Clinical Pathobiochemistry](1999), Page 189) and compared with the blank reading of the medium. Theresults reproduced in FIG. 8 show that the cells have reached themaximum level of insulin production after 14 days in culture. Theinsulin quantities produced by the cells treated in the course of thedifferentiation increased after 14 days to 3 μU/ml, while no humaninsulin was detectable in the control medium. The bars in FIG. 8 eachrepresent three separate values each determined from three independentindividual experiments.

Next to the determination of the insulin production in the deprogrammedstem cells, which were differentiated into insulin producing cellsaccording to the invention, the portion of insulin producing cells weredetermined which still expressed the monocyte-specific surface antigenCD14 also 3 weeks after conducting the dedifferentiation. It was foundthat on a great portion of these cells (about 30 to 40%) themonocyte-specific antigen CD14 was detectable also after 3 weeks.

EXAMPLE 10 Alternative Method for the Production of Hepatocytes fromDedifferentiated Programmable Stem Cells

As an alternative to the use of hepatocyte-conditioned medium (LLCM), asillustrated in Example 7, the differentiation of the stem cells intohepatocytes was induced by the nutrient medium (Ha) indicated below. Theproduction of hepatocytes from stem cells in turn took place in cultureflasks with a volume of approximately 250 ml and flat walls (T75-cellculture flasks). Approximately 5×10⁶ of the cells produced according toExample 13 were introduced into approximately 5 ml of the improvedculture medium indicated below (Ha, differentiation medium forhepatocytes) and after being introduced into the flasks, mixed with afurther 15 ml of culture medium. For the differentiation of the cells,the flasks were incubated in a horizontal position in an incubator at37° C. and 5% CO₂.

Differentiation medium for hepatocytes (Ha) (modified according toSchwarz et al., “Multipotent adult progenitor cells from bone marrowdifferentiate into functional hepatocyte-like cells”, J. Clin. Invest.10 (109), 1291–1302 (2002)):

TABLE 12 RPMI 1640 445 ml Fetal calf serum (FCS)  50 ml Penicillin (100U/l)/Streptomycin (100 μg/l) solution  5 ml Total volume 500 ml

The nutrient medium also contained fibroblast growth factor-4 (FGF-4) ina quantity of 3 ng/ml.

Within the first hour the cells adhere to the bottom of the culturevessel. The differentiation of the stem cells was monitored with regardto albumin production. For this purpose the culture medium was changedat intervals of approximately 2 to 3 days, the cell supernatantcollected each time, and frozen at −20° C. The cells adhering to thebase of the culture flask could be detached by tryptinization asillustrated in Example 2.

The albumin content of the supernatant collected at the different timeswas measured by means of ELISA (Enzyme-linked-immunosorbent-assay) forhuman albumin (according to the protocol of Bethyl Laboratories Inc. andaccording to Schwarz et al., loc. cit.) and compared with the blankreading of the medium. The results presented in FIG. 9 show that thealbumin production of the cells during the period of 14 to 28 days inculture remained approximately constant. The measurements were carriedout on days 0 (blank reading of the medium), 14, 21, 28 and 30 relativeto the time of addition of the Ha medium. The values determined in eachcase amounted to ca. 5 ng/ml, 450 ng/ml, 425 ng/ml, 440 ng/ml and 165ng/ml. The bars in FIG. 9 each represent three separate values eachdetermined from three independent individual experiments.

EXAMPLE 11 Determination of the Co-Expression of Albumin and of theMonocyte-Specific Antigen CD14 in Hepatocytes Derived fromDedifferentiated Stem Cells

The determination of the co-expression of albumin and of themonocyte-specific antigen CD14 in hepatocytes derived fromdedifferentiated stem cells was carried out on the one hand bydouble-staining (A) and on the other hand by FACS analysis (B).

-   A) Stem cells according to the invention differentiated into    hepatocytes according to Example 10 were cultivated on cover glasses    in a 6-well plate and fixed with methanol as illustrated in    Example 4. A double-staining was then carried out, in order to    detect the simultaneous expression of the antigen CD 14 (phenotype    marker of monocytes) on the one hand and of albumin (liver-specific    marker) on the other hand.

For this purpose the cells were first incubated as illustrated inExample 4 with a primary antibody against human albumin (guinea pig vs.human albumin) in a 1:50 dilution in PBS. Following a washing step, thecells were then incubated for 45 minutes with a secondary antibody mouseanti-rat, which binds the guinea pig antibodies, also in a 1:50 dilutionin PBS. The staining process was then carried out according to Example 4using the method of Cordell J. L., et al. (loc. cit.) with APAAP redcomplex.

For the second staining step, the cells were then incubated with theprimary antibody, mouse anti-human-CD14, and following a washing stepaccording to Example 4 stained with the ABC Streptavidin KIT ofVectastain (Vector) using the method of Hsu, S. M., et al. “The use ofantiavidin antibody and avidin-biotin-peroxidase complex inimmunoperoxidase technics” Am. J. Clin. Pathol. 75 (6): 816–821 (1981)with dem DAB-Complex (brown) (Vector Laboratories).

Nucleus counter-staining with haemalaun was then carried out asillustrated in Example 4, followed by embedding in Kaiser's glycerolgelatin.

The results are shown in FIG. 10. The figure shows the expression of theantigen CD14 as brown color, which slowly decreases parallel to themorphological transformation of the cells into hepatocytes, while thealbumin expression as red color increases with the increasing maturationof the hepatocytes. Picture No. 4 in FIG. 10 shows the cells after threeweeks stimulation with the hepatocyte-conditioned medium. B) Parallelwith the double marking, the stem cells differentiated into hepatocytesaccording to the invention were subjected to FACS(fluorescence-activated cell sorting) analysis.

The stem cells differentiated into hepatocytes according to theinvention according to Example 10 were first harvested by mechanicaldetachment of the cells from the culture cell scraper. The cells werecarefully rinsed from the flask with PBS and washed twice, each time in10 ml of PBS-solution. For this purpose the cell suspensions in the PBSsolution were introduced into a 15-ml centrifuge tube and precipitatedat 1600 rpm. The resultant cell sediment was diluted with PBS, such thatexactly 1×10⁵ cells were present in 100 μl PBS.

10 μl of each of FITC-marked anti-CD 14 antibodies (BD Pharmingen) orFITC-marked anti-albumin antibodies (Beckmann) and FTTC-markednon-specific IgGI mouse anti-human antibodies were then added to thiscell suspension. After an incubation period of 20 minutes the cells wereresuspended twice in 500 μl PBS and each precipitated for 5 minutes at1600 rpm and then finally taken up in 200 μl PBS. After resuspension ofthe cells, fluorescence was measured with a BD FACScalibur flowcytometer from the company BD Biosciences (Franklin Lakes, N.J.) (cf.Bruhn H. D., Fölsch U. R. (Eds.), Lehrbuch der Labormedizin: Grundlagen,Diagnostik, Klinik Pathobiochemie [Textbook of Laboratory Medicine,Principles, Diagnosis, Clinical Pathobiochemistry], 395–403 (1999); andHolzer U. et al., “Differential antigen sensitivity and costimulatoryrequirements in human Th1 and Th2 antigen-specific CD4(+) cells withsimilar TCR avidity” J. Immunol. 170 (3): 1218–1223 (2003)). Theevaluation of the results was carried out using the Microsoft WinMDIprogram with reference to Marquez M. G., et al. “Flow cytometricanalysis of intestinal intra-epithelial lymphocytes in a model ofimmunodeficiency in Wistar rats.” Cytometry 41 (2): 115–122 (2000).

The results of the FACS-Analysis are reproduced in FIG. 11. The figureshows the expression of the CD14 (top row) and of the albumin antigen(bottom row), which was measured in dedifferentiated monocytes(left-hand column) and in the stem cells differentiated into hepatocytesaccording to the invention (right-hand column). In dedifferentiatedmonocytes a strong expression of CD14, but no expression of albumincould be detected, while in the hepatocytes developed fromdedifferentiated monocytes a weaker expression of the CD14 and a verystrong expression of the albumin was detectable.

EXAMPLE 12 In Vivo Use of Dedifferentiated Programmed Stem Cells ofMonocytic Origin

In order to clarify, to what extent the programmable stem cells in vivoafter injection via the portal vein into the liver of a geneticallyidentical recipient animal undergo a specific differentiation via thesignal-providers present in the liver, livers of female LEW rats werefirst treated with retrorsine, in order to inhibit the hepatocytespresent in the liver (liver parenchyma cells) regarding theirproliferation activity (Ref. Lacone, E., et al. “Long-term, near-totalliver replacement by transplantation of isolated hepatocytes in ratstreated with retrorsine” Am. J. Path. 153: 319–329 (1998)).

For this purpose the LEW rats received 30 mg of the pyrrolizidinealkaloid retrorsine, injected intraperitoneally twice within 14 days.Subsequently an 80% resection of the livers treated in this way wascarried out, followed by the administration of 5×10⁵ of the programmablestem cells in 1 ml PBS into the portal vein of the remaining residualliver. The stem cells had been obtained, as illustrated in Example 2,from monocytes of male LEW rats. Five days after administration of thestem cells a punch biopsy of the liver was carried out for histologicalassessment of the liver and to detect the cell types differentiated fromthe stem cells by means of fluorescence-in-situ-hybridization (FISH)with Y-chromosome-specific probes, as illustrated in detail in Hoebee,B. et al. “Isolation of rat chromosome-specific paint probes bybivariate flow sorting followed by degenerate oligonucleotideprimed-PCR.” Cytogenet. Cell Genet. 66: 277–282 (1994).

FIG. 7A shows the Y-chromosome-positive (red points in the cell nucleus)hepatocytes derived from the male LEW stem cells on the 5th day afterintraportal injection into retrorsine-pretreated 80%-resectioned liversof female recipient animals. The selective removal of the same liver onday 25 after stem cell injection shows the differentiation of the stemcells into hepatocytes, endothelial cells and bile duct epithelia (FIG.7B). At this point in time, the liver has already reached its normalsize, and >90% of the cells have a Y-chromosome. From this, it can beconcluded, that the injected syngenic programmable stem cells ofmonocytic origin in are capable in vivo, of effecting a completerestoration of the liver with normal metabolic function. FIG. 7C showsin this connection the Kaplan-Meier survival curves (n=4 per group) ofstem-cell-treated versus untreated recipient rats followingadministration of retrorsine and 80% liver resection.

The function parameters bilirubin and ammonia (NH₃) prove the completemetabolic functionality of the long-term surviving stem-cell-treatedanimals (FIGS. 7D and 7E).

EXAMPLE 13 Propagation and Dedifferentiation of Monocytes in CellCulture Flasks

Cultivation and propagation of the monocytes on the one hand and thededifferentiation of the cells of the other side on a larger scale wereconducted in culture flasks in the same nutrient medium, which was alsoused for the cultivation in well-plates (cf. Example 2). The nutrientmedium contains 2.5 μg/500 ml M-CSF and 0.2 μg/500 ml interleukine 3(IL-3).

The monocytes isolated in Example 1 were transferred to the bottom ofculture flasks having a volume of 250 ml and flat walls (T75-cellculture flasks). About 10 times×10⁶ cells were transferred into eachflasks and were each filled up with 20 ml of the above indicatednutrient medium. The determination of this cell number for the exactdosing per flask was carried out according to known procedures, cf. HayR. J., “Cell Quantification and Characterization” in Methods of TissueEngineering, Academic Press (2002), Chapter 4, pages 55–84.

The cell culture flasks were incubated in an incubator at 37° C. for 6days. After 24 hours, the cells settled at the bottom of the flasks. Thesupernatant was removed every second day and the flasks were each filledwith 20 ml fresh nutrient medium.

On day 6, the flasks were rinsed twice with 10 ml PBS each, after thenutrient medium had previously been pipetted off from the flasks.Hereby, all cells were removed, which did not adhere to the bottom ofthe flasks. The cells growing adhere to the bottom of the flasks weresubsequently removed from the bottom of the flasks with a sterile cellscraper. The separated cells were now removed from the flasks by rinsingwith PBS and were pooled in a 50 ml Falcon tube and were centrifuged at1800 rpm for 10 minutes. Thereafter, the supernatant was discarded andthe sediment was resuspended in fresh RPMI 1640 medium (2 ml/10⁵ cells).

This cell suspension could be used directly for differentiating intovarious target cells.

Alternatively, the cells were mixed with DMSO/FCS as freezing mediumafter centrifugation and were deep-frozen at a concentration of 10⁶/ml.

The freezing medium contained 95% FCS and 5% DMSO. About 10⁶ cells weretaken up in 1 ml of the medium and were cooled following the subsequentsteps:

30 minutes on ice;

2 hours at −20° C. in precooled styropor box;

24 hours at −80° C. in styropor;

stored in tubes in liquid nitrogen (N₂) at −180° C.

1. A dedifferentiated, programmable cell of human monocytic origin,wherein said dedifferentiated, programmable cell of human monocyticorigin expresses a CD14 antigen, a CD90 antigen, and a CD123 antigen. 2.The dedifferentiated, programmable cell of human monocytic origin ofclaim 1, further comprising a transfected gene.
 3. A dedifferentiated,programmable cell of human monocytic origin, wherein saiddedifferentiated, programmable cell of human monocytic origin expressesa CD14 antigen, a CD90 antigen, and a CD135 antigen.
 4. Thededifferentiated, programmable cell of human monocytic origin of claim3, further comprising a transfected gene.
 5. A dedifferentiated,programmable cell of human monocytic origin, wherein saiddedifferentiated, programmable cell of human monocytic origin expressesa CD14 antigen, a CD123 antigen, a CD90 antigen, and a CD135 antigen. 6.The dedifferentiated, programmable cell of human monocytic origin ofclaim 5, further comprising a transfected gene.
 7. A pharmaceuticalcomposition comprising a dedifferentiated, programmable cell of humanmonocytic origin, wherein said dedifferentiated, programmable cell ofhuman monocytic origin expresses a CD14 antigen and a CD90 antigen.
 8. Apharmaceutical composition comprising a dedifferentiated, programmablecell of human monocytic origin, wherein said dedifferentiated,programmable cell of human monocytic origin expresses a CD14 antigen, aCD90 antigen, and a CD123 antigen.
 9. A pharmaceutical compositioncomprising a dedifferentiated, programmable cell of human monocyticorigin, wherein said dedifferentiated, programmable cell of humanmonocytic origin expresses a CD14 antigen, a CD123 antigen, a CD90antigen, and a CD135 antigen.
 10. A dedifferentiated, programmable cellof human monocytic origin, wherein said dedifferentiated, programmablecell of human monocytic origin expresses a membrane associatedmonocyte-specific surface antigen, CD14 and a CD90 antigen.
 11. Thededifferentiated, programmable cell of human monocytic origin accordingto claim 10, wherein said dedifferentiated, programmable cell of humanmonocytic origin is transfected with one or more genes.
 12. Apreparation of a dedifferentiated, programmable cell of human monocyticorigin according to claim 10, wherein said preparation is in a suitablemedium.
 13. A dedifferentiated, programmable cell of human monocyticorigin, wherein said dedifferentiated, programmable cell of humanmonocytic origin expresses a CD14 antigen, a CD90 antigen, and has acapacity to differentiate into a cell type selected from the groupconsisting of neurons, endothelial cells, adipocytes, hepatocytes,keratinocytes, and insulin-producing cells.
 14. A dedifferentiated,programmable cell of human monocytic origin, wherein saiddedifferentiated, programmable cell of human monocytic origin expressesa CD14 antigen a CD90 antigen, and has a capacity to differentiate intoany cell type from the group consisting of glia cells, neurons, neuronalprecursor cells, endothelial cells, adipocytes, hepatocytes,keratinocytes, and insulin-producing cells.